Lysosomal proteinases are translated as preproenzymes, transferred through the Golgi apparatus as proenzymes, and localized in lysosomes as the mature enzymes. Pulse-chase analyses and the immunoisolation of proenzymes or recombinant proenzymes are useful tools for analyzing this process, but the processing proteinases that participate in this pathway are largely unknown. Recently, we developed a new method for analyzing processing proteinases using Bafilomycin A1 and proteinase inhibitors. Here we summarize the recent progress including our results obtained using this method.