Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation <15%). This improved protocol is fast and reproducible. Its linear range of almost two log scales permits assay of proteolytic activity in a wider range than current methods.