Ostroh Academy - the first higher educational institution in Ukraine and Eastern Europe - was founded in 1576 by Prince Vasily-Konstantin Ostrozhsky and Princess Galshka Ostrozhskaya. Slavic, Greek, Latin and the so-called liberal sciences (grammar, arithmetic, rhetoric, logic, etc.) were taught at this school. Its first rector was the writer G.D. Smotrytsky, and the pupils were the famous scientist and writer M. Smotrytsky, hetman Petro Sahaydachny, Demyan Nalivaiko (brother of Severin Nalivaiko) and others.

In the thousand-year history of Ukrainian culture, Ostrog played the role of an intellectual center at the turn of the 16th-17th centuries, and the academy, which operated here in 1576-1636, was the leading scientific institution in Ukraine. Her printing house became famous for the world's first edition of the Bible in Church Slavonic (1581).
The founder of the academy, Vasily-Konstantin Konstantinovich Ostrozhsky, was one of the richest magnates of the state, a philanthropist. As S. Kardashevich notes, the prince was three times as wealthy as the king of the Commonwealth. He owned a third of all Volyn, estates in Kiev, Vladimir, Belotserkovsky, Boguslavsky, Pereyaslavsky, Kanevsky and Cherkassy elderships. The prince's possessions included more than 3,040 cities and villages, trading houses in Gdansk, Yaroslav, Lvov, castles in Dubno, Turov, Tarnow, courtyards-chambers in Warsaw and Vilna.

Three periods can be clearly distinguished in the history of the academy. The first of these is the time of formation (1576-1586). It is characterized by powerful intellectual flash. Already in the first years of its existence, the academy becomes a unique cultural and educational center with a publishing house created in it. Figures of different faiths were involved in the work in the institution - Gerasim Smotrytsky, Ivan Fedorov, the Greeks Immanuel Moshopoulos, Dionysius Ralli and Eustachius Nathanael, Simon Pekalid, Belarusian Protestants Andrei Rymsha and Ukrainian Motovilo.
The next period of the academy's activity (1587–1620) is characterized by its highest flourishing. Outstanding personalities arrived in Ostrog - Ukrainians and foreigners with rich European scientific and pedagogical experience. Ukrainian Kupriyan studied in Padua and Venice, Isakiy Boriskovich - in Alexandria. Among the pupils of the Padua Academy, the Greeks Nikifor Kantakouzin and Cyril Loukaris stood out. Mutually beneficial cultural ties with the monasteries of Athos were established as early as the 70s of the 16th century. They were visited by Ivan Vishensky, Isakiy Boriskovich. At the same time, Athonite monks came to Ostrog for local publications, supplying the printing house with the necessary manuscripts.
Polish leaders actively cooperated with the academy. One of them was Jan Latosh, an astronomer, mathematician, doctor of medicine, a graduate of the University of Padua and Krakow. Self-taught Ukrainians, the recognized Orthodox publicist Gerasim Smotrytsky, the first rector of the academy of the same name, polemist Ivan Vishensky, writer Demyan Nalivaiko, his brother, leader of the popular uprising in Ukraine Severin Nalivaiko, brought glory to Ostrog. The atmosphere that prevailed in Ostrog at the turn of the two centuries played a decisive role in the rapid activity of the academy: different national and cultural traditions coexisted here. In a city with a population of five thousand people, with the help of Italian architects in the Renaissance style, a castle, three wooden palaces, a town hall, and city towers were built. There were a church, two synagogues with a well-known yeshiba - a spiritual and general education school, a Tatar mosque, Calvinist and Protestant churches.
The decline of the academy, and later its liquidation, turned out to be the result of the activities of the Jesuits. They sought to strengthen their role in this region, and this was impossible without the liquidation of the cultural and ideological center. After all, the Ostroh elite, with their active Orthodox propaganda, prevented the implementation of the far-reaching intentions of the Vatican and the Polish gentry. Since the Jesuits failed to persuade Prince Ostrozhsky to their side, they directed all their energy to the prince's heirs. His eldest son Janusz became a Catholic, and in 1583 his second son, Konstantin, also changed his faith. Another son, Alexander, in 1592 marries the well-known patroness of the Jesuits, Anna Kostchanka. In 1636, a gentry court was organized over the Ostrozhans, they were tortured and executed. The court introduced a union in Ostrozhchina. The greatest manifestation of the fanaticism of Anna-Aloise Ostrozhskaya was her will (1654), according to which numerous estates and large sums of money became the property of the protected Jesuit order, churches, and Catholic hospitals.