An in vitro crustacean (freshwater shrimp,Macrobrachium potiuna) erythrophore bioassay for chromatophorotropins and other pigment cell agonists is described. The present assay is a quantitative method that determines the pigment responses with the aid of an ocular micrometer. The pigment granules within the erythrophores are dispersed out into the dendritic processes of the cells when the isolated carapace is placed in physiological solution. This bioassay provides, therefore, a method for measuring the response of the pigment cells to aggregating agents such as pigment concentrating hormone (PCH). This bioassay is sensitive to PCH at a concentration as low as 3 ± 10−12M. Calcium ionophore A23187 mimics the actions of PCH, but, unlike the hormone, the Ionophore‐induced pigment aggregation is irreversible after physiological solution rinses. Therefore, chromatophorotropic activities of pigment dispersing agents, such as pigment dispersing hormones (PDH), can be determined on ionophore‐treated erythrophores. The potencies of α‐PDH and β‐PDH show a threefold difference (not significant). Because of its convenience and its ability to make an objective determination of the bidirectional pigment movements within erythrophores, this bioassay is a suitable method for further structure‐activity studies of the various chromatophorotropins and