We developed phospho-ERK1/2 ELISA for human and rainbow trout liver cells, employing HepG2 and RTL-W1 cell lines as models. The assay was applied to detect changes in ERK1/2 activity for nine chemicals, added over a wide concentration range and time points. Cell viability was measured to separate ERK1/2 regulation from cytotoxicity. Perfluorooctane sulfonate and carbendazim did not change ERK1/2 activity; influence on ERK1/2 due to cytotoxicity was indicated for tributyltin and cypermethrin. Mancozeb, benzo[a]pyrene, and bisphenol A stimulated ERK1/2 up to ∼2- (HepG2) and 1.5 (RTL-W1)-fold, though the kinetics differed between chemicals and cell lines. Bisphenol A and benzo[a]pyrene were the most potent concentration-wise, altering ERK1/2 activity in pM (HepG2) to nM (RTL-W1) range. While atrazine and ibuprofen increased ERK1/2 activity by ∼2-fold in HepG2, they did not initiate an appreciable response in RTL-W1. This assay proved to be a sensitive, medium- to high-throughput tool for detecting unrecognized ERK1/2-disrupting chemicals.