This chapter presents the procedure for purification and assaying of crab collagenolytic protease. The procedure described in the chapter uses the glands from 8000 crabs, received in shipments of 1000 each. The purification steps are following: preparation of acetone powder; first gel filtration; ion-exchange chromatography; hydroxyapatite chromatography; second gel filtration. This procedure produces approximately 100-150 mg of homogeneous crab protease from the glands of 8000 fiddler crabs. The enzyme is stable for several months in slightly acidic solution (pH -6.0) at –20°C, and can be stored indefinitely as a lyophilized powder. Crab collagenase can be assayed specifically for collagenolytic activity with a standard assay, which quantitates the release of soluble [14C]glycine-containing peptides from native reconstituted guinea pig skin collagen fibrils, or spectrophotometrically by following esterase or amidase activity with synthetic substrates.