tFor years, d-amino acids were thought to have a minor function in biological processes compared to thatof l-enantiomers. Recently, many studies have shown that d-amino acids are present in high concentra-tions in microorganisms, plants, mammals and humans and execute specific biological functions. Onerelevant example is that of d-cysteine, whose hydrogen sulfide-producing properties have been found toprotect neurons against oxidative stress and to promote dendritic development. Herein, we introduce achiral LC MS method for the rapid determination of cysteine enantiomers under polar ionic elution con-ditions (MeOH/MeCN/H2O 49/49/2 v/v/v, containing 50 mM formic acid and 50 mM ammonium formate)developed on a Chiralpak®ZWIX(+) chiral stationary phase. Cysteine enantiomers were analysed in bio-logical samples after efficient reduction of the disulfide bond in cystine; the latter was achieved with theuse of 1,4-dithio-dl-threitol as a reducing agent. A baseline resolution (RS= 2.7) was obtained, and thed-enantiomer eluted before the l-enantiomer. For the enantioselective analysis, cysteine was labelledwith AccQ-Tag reagent, resulting in improved chromatographic behaviour and MS detection sensitivity.The method was validated according to the Food and Drug Administration guidelines. Good linearity wasdetermined in the ranges of 0.05-0.50 mg/L for d-cysteine and 0.11-0.56 mg/L for l-cysteine. The repeata-bility and intermediate precision were found to be lower than 4.0%, with trueness ranging from 95.6 to100.2% for both enantiomers. The LOD and LOQ values were 0.02 and 0.05 mg/L for d-cysteine and 0.04and 0.11 mg/L for l-cysteine, respectively. The method was successfully applied to cell culture samplestreated with d-cysteine.