Individuals aboard jet aircraft may be exposed to potentially toxic triaryl organophosphate anti-wear lubricant additives (TAPs) that are converted by cytochromes P450 into toxic metabolites. Consequences of exposure could be reduced by using less toxic TAPs. Our goal was to determine whether an in vitro assay for inhibition of butyrylcholinesterase (BChE) by bioactivated TAPs would be predictive of inhibition of serine active-site enzymes in vivo. The in vitro assay involved TAP bioactivation with liver microsomes and NADPH, followed by incubation with human BChE and measurement of BChE activity. Of 19 TAPs tested, tert-butylated isomers produced the least BChE inhibition. To determine the relevance of these results in vivo, mice were exposed to Durad 125 (D125; a commercial mixture of TAP esters) or to TAPs demonstrating low or no BChE inhibition when assayed in vitro. Inhibition of BChE by bioactivated TAPs in vitro correlated well with inhibition of other serine active-site enzymes in vivo, with the exception of brain acetylcholinesterase and neuropathy target esterase (NTE), which were not inhibited by any TAP tested following single exposures. A recombinant catalytic domain of NTE (rNEST) exhibited classical kinetic properties of NTE. The metabolite of tri-(o-cresyl) phosphate (ToCP), 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP), inhibited rNEST in vitro, but with an IC(50) value almost 6-times higher than for inhibition of BChE. Physiologically-relevant concentrations of the flavonoid naringenin dramatically reduced D125 bioconversion in vitro. The in vitro assay should provide a valuable tool for prescreening candidate TAP anti-wear additives, identifying safer additives and reducing the number of animals required for in vivo toxicity testing.