Osthoff studied classical philology, Germanic philology, Sanskrit and comparative linguistics in Berlin, Tübingen and Bonn. In 1869 he obtained his doctorate in Bonn as a student of Hermann Usener. During his time in that city he became a member of the Burschenschaft Alemannia of Bonn.
Osthoff's academic journey involved studying classical philology, Germanic philology, Sanskrit, and comparative linguistics. He received his doctorate from the University of Bonn under the guidance of Hermann Usener in 1869. As a member of the Burschenschaft Alemannia, he was part of a student organization at the University of Bonn.