Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for the catalytic hydrolysis or stoichiometric binding of organophosphates (OPs). Previously, rationally designed hBChE mutants (G117H and E197Q) were reported in the literature and showed the feasibility of engineering OP hydrolytic functional activity into hBChE. However, the OP hydrolysis rate for G117H is too low for clinical utility. Additional OP-resistant hBChE variants with greater hydrolysis rates are needed as OP nerve-agent countermeasures for therapeutic utility. As described herein, a directed molecular evolution process was used to identify amino acid residues that contribute to OP-resistant functional activity of hBChE variants. In this article, we describe the development and validation of a novel method to identify hBChE variants with OP-resistant functional activity (decreased rate of OP inhibition). The method reported herein used an adenoviral protein expression system combined with a functional screening protocol of OP nerve-agent model compounds that have been shown to have functional properties similar to authentic OP nerve-agent compounds. The hBChE screening method was robust for transfection efficiency, library diversity, and reproducibility of positive signals. The screening approach not only identified the previously reported hBChE G117H variant, but also identified a series of additional hBChE variants, including hBChE G117N, G117R, E197C, and L125V, that exhibited OP-resistant functional activities not reported previously. The mammalian functional screening approach can serve as a cornerstone for further optimization and screening for OP-resistant hBChEs for potential therapeutic applications.