The development of drugs with the ability to increase the level of the antioxidant glutathione and related metabolites has become an important research area for many age-related diseases. Here we describe a high-performance liquid chromatography (HPLC) method that uses the thiol-specific, fluorogenic reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) for the simultaneous determination of total glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), and homocysteine (Hcys) in cell culture medium. ABD-F-labeled thiols were separated using an isocratic mobile phase consisting of 14% methanol and 86% 0.1 M acetate buffer at pH 4.0. The method was validated for linearity, accuracy, and intra- and interday precision, and the lower and upper limits of quantitation (LLOQ and ULOQ, respectively) were determined using a Dionex RF-2000 detector set to medium sensitivity. In addition, the suitability of N-acetylcysteine (NAC) as an internal standard was evaluated by external and internal standard calibration methods. Although both calibration methods showed acceptable linearity (correlation coefficients > 0.99) and intra- and interday precision (relative standard deviations = 10.2 and 6.6%, respectively), the external standard calibration method performed better in terms of accuracy (recovery = 93.7–125%) and also had lower LLOQ values for all analytes (Cys = 6.3 μM, CysGly = 0.8 μM, Hcys = 0.8 μM, and GSH = 1.6 μM).