Cathepsin L is a lysosomal protease that can be secreted from the cell and is implicated to play a role in protein degradation, arthritis, apoptosis, and cancer. TheProcathepsin L can be auto-catalytically activated to mature Cathepsin L (~24 kDa) by adding an equal volume of 0.2 M NaOAc, 5 mM EDTA pH 4.0 containing fresh 5 mM DTT at RT. The enzyme is fully activated within 30 min of incubation at RT. After activation mature Cathepsin L is highly auto-proteolytic at pH 4.0, and care must be taken to avoid self-proteolysis. If the activated enzyme is not used immediately, methylmethanthiosulfonate (1mM final concentration; MeS-SO2Me; MMTS) could be added and sample should be frozen at -80C. The activity can be then restored by adding L-cysteine (3 M excess over MMTS) to the enzyme solution.