The single-cell gel electrophoresis (SCGE), or comet assay, is a simple method that uses microgel and electrophoresis to evaluate damage in the DNA of the cells. The first protocol adapted for plants was described in the 1990s, however, a universal standardized procedure is not available to date. This study aimed to evaluate the influence of exposure time in the comet assay in plant models Allium cepa L. and Lactuca sativa L. Root tips of both plant models were exposed to methyl methanesulfonate (MMS), a known genotoxicant, for 2 h to 36 h. The alkaline version of the comet assay for plant samples was performed, and the DNA damage was assessed. Arbitrary units (AU) were calculated and compared to the negative control. MMS induced DNA damage (P < 0.05) after 2, 6, 10, 14, 28, 32 and 36 h of exposure in A. cepa, and at 2, 8, 10, 14 and 36 h for L. sativa. The highest AU values for A. cepa were observed after 4 h of exposure to MMS and for L. sativa, after 24 h of exposure. The optimal exposure times considering both genotoxic and mutagenic risk were indicated.