In 1612 he entered the University of Jena. In 1615, with the idea of studying law, he moved to Wittenberg. In consequence of an illness, however, he returned to Jena after a year. Here, as a student of theology under Johann Gerhard, he directed his attention especially to Hebrew and the cognate dialects; in 1619 he was made an adjunctus of the philosophical faculty, and some time afterwards he received an appointment to the chair of Hebrew.
From 1625 to 1638 he was superintendent in Sondershausen; but shortly after the death of Gerhard (1637) he was, in accordance with Gerhard's last wish, appointed to succeed him at Jena. In 1640, however, at the earnest invitation of Duke Ernest the Pious, he removed to Gotha as court preacher and general superintendent in the execution of important reforms which had been initiated in the ecclesiastical and educational establishments of the Duchy. The delicate duties attached to this office he discharged with tact and energy; and in the Syncretistic Controversy, by which Protestant Germany was so long vexed, he showed an unusual combination of firmness with liberality, of loyalty to the past with a just regard to the demands of the present and the future.
His principal work, Philologia sacra (1623), marks the transition from the earlier views on questions of biblical criticism to those of the school of Spener. It was more than once reprinted during his lifetime, and appeared in a new and revised form, edited by J. A. Dathe (1731-1791) and G. L. Bauer at Leipzig. Glassius succeeded Gerhard as editor of the Weimar Bibelwerk, and wrote the commentary on the poetical books of the Old Testament for that publication. A volume of his Opuscula was printed at Leiden in 1700.