Abstract The aim of this study was to evaluate melatonin cytotoxicity by measuring its effects on various cellular targets. Cell viability, intracellular reduced glutathione (GSH) level, and reactive oxygen species (ROS) production were assessed in the human liver cell line (HepG2), after incubation with increasing melatonin concentrations (0.1–10,000 μM). The incubation times tested were 24, 72, and 96 h for cell viability and intracellular GSH level, and 15 and 45 minutes for ROS production. Cellular target evaluations were possible in living cells by means of a new microplate cytofluorimeter. This technology was suitable for the assessment of cell viability, GSH level, and ROS overproduction with, respectively, neutral red, monochlorobimane (mBCl), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescent probes. At the lowest melatonin concentrations (0.1–10 μM) and for a relatively short incubation time (24 h), the antioxidant effect of melatonin was revealed by an increased intracellular GSH level, associated to cell viability improvement. In contrast, after longer incubation (96 h), cell viability significantly decreased with these lowest melatonin concentrations (0.1–10 μM). Moreover, high melatonin concentrations (1,000–10,000 μM) induced GSH depletion. This oxidative stress is associated with ROS overproduction from 10 μM after only 15 minutes of incubation. This dual effect is strong evidence that, in vitro, melatonin can be both antioxidant and prooxidant on the human liver cell line, depending on the concentration and incubation time.