Display omitted ► A new, rapid and sensitive on-line HPLC assay for biothiols was proposed. ► Postcolumn derivatization was performed with methyl-β-cyclodextrin-containing DTNB. ► Biothiols were selectively determined individually among other antioxidants. ► The oligosaccharide helped to improve the sensitivity. A novel on-line HPLC-DTNB method was developed for the selective determination of biologically important thiols (biothiols) such as l-cysteine (Cys), glutathione (GSH), homocysteine (HCys), N-acetylcysteine (NAC), and 1,4-dithioerythritol (DTE) in pharmaceuticals and tissue homogenates. The biothiols were separated on C18 column using gradient elution, reacted with the postcolumn reagent, DTNB in 0.5% M-β-CD (w/v) solution at pH 8, to form yellow-colored 5-thio-2-nitrobenzoic acid (TNB), and monitored with a PDA detector (λ=410nm). With the optimized conditions for chromatography and the post-column derivatization, 40nM of NAC, 40nM of Cys, and 50nM of GSH can be determined. The relative standard deviations of the recommended method were in the range of 3.2–5.4% for 50μM biothiols. The negative peaks of biothiol constituents were monitored by measuring the increase in absorbance due to TNB chromophore. The detection limits of biothiols at 410nm (in the range of 0.04–0.58μM) after post-column derivatization with DTNB+M-β-CD were much lower than those at 205nm UV-detection without derivatization, and were distinctly lower than those with post-column DTNB alone. The method is rapid, inexpensive, versatile, nonlaborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of biothiol constituents of biological fluids and pharmaceuticals.