We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.