Abstract We report the development of an enzyme-linked immunosorbent assay (ELISA) for protein tyrosine phosphatases (PTPases). PTPase activitym was monitored by quantitating disappearance of O- phospho- l-tyrosine (P-Tyr) in an ELISA system using antigen captured followed by double antibody labelling. PTPase activity of agarose conjugated PTB-1B was demonstrated using the ELISA system. PTPase activity was sensitive to both PTB-1B concentrations and time of incubation. 1 mU of PTPase activity was defined as that amount of enzyme producing a rate of loss of 0.01 absorbance units/minute with a specific activity of 150 pmol P-Tyr/min per μg protein based on the unit of PTPase activity from the conventional assay system. The PTP-1B activity was shown by the ELISA system to be completely inhibitable by Poly (Glu,Tyr)4: 1 at 100 μg/ml. We used the ELISA system to detect PTPase activity in lysates of cultured cells. The PTPase activity cell lysates of MDA-MB 468 breast carcinoma cells as obtained by the ELISA were compared with those obtained by a standard 32P i release assay using radio-labelled Raytide tm as PTPase substrate. The decrease in P-Tyr concentration was dependent on the time of incubation with the lysate and on lysate concentration and compared well with the release of 32P i in the radioactive assay system. Orthovanadate as well as heat denaturation inhibited the PTPase activity of the cell lysates in both the assay systems. The assay presented here is a simple immunological system capable of measuring activity of purified PTPase as well as PTPase levels in cell and tissue extracts.