PIWI proteins, a conserved subfamily of Argonaute proteins that interact with PIWI-interacting RNAs (piRNAs), have been implicated in the pathogenesis of several cancers, including glioblastoma (GBM). This project investigates the expression patterns of the four human PIWI proteins in GBM tissues and glioblastoma stem cells (GSCs) – a therapy-resistant cell population that contributes to tumor aggressiveness and recurrence. We conducted a comprehensive analysis of PIWI expression in GBM. Our results identify PIWIL2 as the predominant PIWI protein significantly enriched in GSCs. Moreover, we report the first evidence of PIWIL4 expression in GBM and question the previously assumed roles of PIWIL1 and PIWIL3 in this context. The deposited data represent mass spectrometry analysis of paired GSCs and non-stem cells derived from human GBMs. In addition, it contains data from GSCs differentiated using 10% fetal calf serum for at least 10 days.