Most luminal lysosomal proteins are synthesized as precursors containing mannose 6-phosphate (Man6-P) and a number of recent studies have conducted affinity purification of Man6-P containing proteins as a step toward defining the composition of the lysosome. Approximately 60 known lysosomal proteins have been found in such studies as well as many other Man-6-P glycoproteins, some of which represent new lysosomal proteins. The latter are of considerable interest from cell-biological and biomedical perspectives, but differentiating between them and other proteins remains a significant challenge. The aim of this study was to conduct a global analysis of the mammalian Man6-P glycoproteome, implementing technical and biostatistical methods to aid in the discovery and validation of lysosomal candidates. We purified Man6-P glycoproteins from 17 individual rat tissues. To distinguish nonspecific contaminants (i.e., abundant or "sticky" proteins that are not fully removed during purification) from specifically purified proteins, we conducted a semiquantitative mass spectrometric comparison of protein levels in nonspecific mock eluates versus specific affinity chromatography eluates to identify those proteins that are specifically purified. We identified 60 known lysosomal proteins, representing nearly all that are currently known to contain Man-6-P. We also find 136 other proteins that are specifically purified but which are not known to have lysosomal function. This approach provides a list of candidate lysosomal proteins and also provides insights into the relative distribution of Man6-P glycoproteins.