Updated project metadata. The differentiation from neural stem cells (NSCs) to oligodendrocyte precursor cells (OPCs) is only incompletely understood. In the current study, we used the so-called neurosphere assay to study this process. We performed in vitro differentiation from neurospheres (which predominantly consist of NSCs) to oligospheres (consisting mainly of OPCs) and investigated their proteome by 6plex TMT-labeling and their phosphoproteome by 3plex dimethyl labeling. Among others, we identified a continuous upregulation of double cortin like kinase 1 (Dclk1) as well as its phosphorylation sites in the so-called SP-rich region. Using western blotting and qPCR, we show that two different Dclk1 isoforms (long and short) are present in NSCs and OPCs which gradually interchange during the investigated differentiation process. We further demonstrate that Dclk1 long is proteolytically processed into Dclk1 short and that this process is regulated via phosphorylation in the SP-rich region. Finally, we generated different BioID constructs fused to individual Dclk1 domains and investigate their interactome as well as potential substrates of Dclk1.