Background: Reduced glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions. To study GSH content in human neuronal cell cultures, thiol‐sensitive fluorescent techniques requiring a small number of cells may be of great value, but their GSH specificity has not been established in these cells.Methods: We tested the efficiency of four currently available GSH fluorescent stains in human neurons and SH‐SY5Y neuroblastoma cells, both cultured in microwells, by using a fluorescence plate reader. Cultures were treated with the inhibitor of the GSH synthesis, buthionine sulfoximine (BSO), and progressive GSH depletion was assayed with monochlorobimane (mBCl), monobromobimane (mBBr), 5‐chloromethylfluorescein diacetate (CMFDA), and 7‐amino‐4‐chloromethylcoumarin (CMAC). GSH was also determined by a biochemical method in cell homogenates to obtain quantitative reference values.Results: Neurons and SH‐SY5Y neuroblastoma had basal GSH contents of 27.1 ± 3.2 and 14.5 ± 1.7 nmol/mg protein (n = 5), respectively. An approximate 90% depletion of GSH was obtained after 3 days of exposure to 1,000 μM of BSO in neurons and after 1 day in SH‐SY5Y cells. Cell death through an apoptotic pathway appeared 1–2 days after total GSH depletion. The assayed stains had different degrees of background fluorescence and sensitivity to GSH content, with similar results in both neuronal cell types. The probes mBCl and CMAC showed the lowest background, and the GSH‐depletion curves were most similar to that of the reference method.Conclusions: Both mBCl and CMAC are useful fluorescent stains to determine semiquantitative GSH concentration in human neuronal cell cultures. Cytometry Part A 51A:16–25, 2003.