Microsomal cysteine-S-conjugate (\boldsymbol{N})-acetyltransferase, an enzyme specific for (\mathbf{S})-substituted cysteines, plays an important role in the detoxicative metabolism of xenobiotics by catalyzing the (\mathbf{N})-acetylation of cysteine-S-conjugates. Cysteine-S-conjugate (\boldsymbol{N})-acetyltransferase activity is generally assayed by measuring the amount of (\mathrm{N}-\left[{ }^{14} \mathrm{C}\right]) acetyl-S-benzyl-L-cysteine generated from the model compound (S)-benzyl-L-cysteine and (\left[{ }^{14} \mathrm{C}\right]) acetyl-CoA and subsequent extraction of the product. Although sensitive, this method is costly and time consuming. For safety and environmental reasons we developed a nonradioactive assay for cysteine-Sconjugate (\boldsymbol{N})-acetyltransferase activity. Our method depends upon the acetylation of the uv-sensitive model compound 4-nitro-S-benzyl-L-cysteine. The test mixture is separated by HPLC, guaranteeing that no byproducts interfere with the determination of product formation. Radioactive and nonradioactive methods were compared using different porcine kidney samples. With the nonradioactive test we determined values of (K_{\mathrm{m}}) and (V_{\max }) of both 4-nitro-S-benzyl-L-cysteine and acetyl-CoA. In summary, this new nonradioactive assay is sensitive, less costly, safer, less time-consuming, and less laborious than radioactive assays for cysteine(\boldsymbol{S})-conjugate (\boldsymbol{N})-acetyltransferase. (1994 Academic Press, Inc.