A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-delta). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.