Liver cancer is a prevalent form of cancer worldwide. While research has shown that increasing sphingomyelin (SM) hydrolysis by activating the cell surface membrane-associated neutral sphingomyelinase 2 (nSMase2) can control cell proliferation and apoptosis, the role of total glutathione depletion in inducing tumor cell apoptosis via nSMase2 activation is still under investigation. Conversely, glutathione-mediated inhibition of reactive oxygen species (ROS) accumulation is necessary for the enzymatic activity of nSMase1 and nSMase3, increased ceramide levels, and cell apoptosis. This study evaluated the effects of depleting total glutathione in HepG2 cells using buthionine sulfoximine (BSO). The study assessed nSMases RNA levels and activities, intracellular ceramide levels, and cell proliferation using RT-qPCR, Amplex red neutral sphingomyelinase fluorescence assay, and colorimetric assays, respectively. The results indicated a lack of nSMase2 mRNA expression in treated and untreated HepG2 cells. Depletion of total glutathione resulted in a significant increase in mRNA levels but a dramatic reduction in the enzymatic activity of nSMase1 and nSMase3, a rise in ROS levels, a decrease in intracellular levels of ceramide, and an increase in cell proliferation. These findings suggest that total glutathione depletion may exacerbate liver cancer (HCC) and not support using total glutathione-depleting agents in HCC management. It is important to note that these results are limited to HepG2 cells, and further studies are necessary to determine if these effects will also occur in other cell lines. Additional research is necessary to explore the role of total glutathione depletion in inducing tumor cell apoptosis.