An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the crosslinked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP•Mg²⁺, and detecting peptide phosphorylation using the phosphoepitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC₅₀ value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.