Alexander disease (AxD) is a fatal neurodegenerative disorder characterized by the presence in astrocytes of protein aggregates called Rosenthal fibers (RFs). In this work, we used a new biochemical fractionation method to enrich for RFs followed by analysis of this fraction using quantitative iTRAQ mass spectrometry. This work identified 77 proteins not previously associated with RFs. Multiple proteins selected for follow-up were confirmed by immunobloting to be enriched in the RF fraction of both the AxD mouse models and human AxD patients including receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X.