Lysosomes are the main degradative organelles of cells and involved in a variety of processes including the recycling of macromolecules, storage of compounds, and metabolic signaling. Despite an increasing interest in the proteomic analysis of lysosomes, no systematic study of sample preparation protocols for lysosome enriched fractions has been performed to date. In the current study, we used samples enriched for lysosomes by paramagnetic nanoparticles and systematically evaluated experimental parameters for the analysis of the lysosomal proteome. This includes different approaches for the concentration of lysosome-containing fractions; desalting of samples by solid phase extraction; fractionation of peptide samples; and different gradient lengths for LC-MS/MS analyses of unfractionated samples by data dependent and data independent acquisition. Furthermore, we evaluated four different digestion methods including filter aided sample preparation (FASP), in-gel digestion, and in-solution digestion using either RapiGest or urea. Using the combined data, we generated a benchmark lysosomal proteome data set for mouse embryonic fibroblasts as well as a spectral library for the analysis of lysosomes by data independent acquisition.