From 1850 to 1857 Schleicher taught classical philology and the comparative study of Greek and Latin at the University of Prague. During this period he turned to the study of Slavic languages. In 1852 he began research on Lithuanian while living among the peasantry of Prussian Lithuania. This was the first attempt to study an Indo-European language directly from speech rather than from texts. His results appeared in the remarkable Handbuch der litauischen Sprache (1856–57; “Handbook of the Lithuanian Language”), the first scientific description and analysis of Lithuanian, complete with a grammar, reader, and glossary.

In the course of his professorship at the University of Jena (1857–68), he published many works, including the one on which his fame rests, Compendium der vergleichenden Grammatik der indogermanischen Sprachen (1861–62; partial trans., A Compendium of the Comparative Grammar of the Indo-European, Sanskrit, Greek and Latin Languages, 1874–77), in which he studied the common characteristics of the languages and attempted to reconstruct the proto-Indo-European parent language, or Ursprache. Schleicher believed that language is an organism exhibiting periods of development, maturity, and decline. As such, it could be studied by the methods of natural science. Developing a system of language classification resembling a botanical taxonomy, he traced groups of related languages and arranged them into a genealogical tree. His model came to be known as the Stammbaumtheorie, or family-tree theory, and was a major development in the history of Indo-European studies or, more generally, in historical linguistic theory.