A chromophoric bifunctional probe design that allows selective chiroptical sensing of cysteine in aqueous solution is introduced. The common need for chiral HPLC separation is eliminated which expedites and simplifies the sample analysis while reducing solvent waste. Screening of the reaction between six phenacyl bromides and the enantiomers of cysteine showed that cyclization to an unsaturated thiomorpholine scaffold coincides with characteristic UV and CD effects, in particular when the reagent carries a proximate auxochromic nitro group. The UV changes and CD inductions were successfully used for determination of the absolute configuration, enantiomeric composition and total concentration of 18 test samples. This assay is highly selective for free cysteine while other amino acids, cysteine derived small peptides and biothiols do not interfere with the chiroptical signal generation.