The US Environmental Protection Agency is increasingly employing new approach methods (NAMs), including in vitro plasma binding and hepatocyte clearance experiments to collect chemical-species specific data. This paper presents data from plasma binding experiments using rapid equilibrium dialysis (RED) devices and plasma from humans, rats, and rainbow trout with a 4-h incubation time. A total of 54 chemicals, utilizing two concentrations, were tested across the three species resulting in 238 chemical-species specific datasets. Mass balance controls for chemical plasma stability and dialysis system recovery were used to evaluate the datasets and almost 40 % of the datasets (92/238 datasets) produced quantitative measurements. Cross-species comparisons and evaluations of the impact of physicochemical properties on chemical-assay performance were also evaluated. Comparisons of human-rat plasma binding revealed rat plasma generally demonstrated higher f