tert-Butylhydroquinone (TBHQ) losses and the residual levels of 2-tert-butyl-1,4-benzoquinone (TBBQ) in tripalmitin at different heating temperatures with or without reflux over various time intervals were investigated. Heating at 120?°C resulted in the slowest TBHQ loss and the highest TBBQ levels (52.61-62.93?μg/mL). The highest TBBQ concentrations (111.73-164.67?μg/mL) at 5 and 8?h and residual concentrations of 10.23-46.95?μg/mL during heating at 170?°C over 24?h were observed. Furthermore, the potential cytotoxicity of TBBQ to RAW 264.7 cells was evaluated with the MTT assay, Hoechst 33258 staining test, and flow cytometry analysis. Results indicate that TBBQ dose- and time-dependently decreased the growth of cells and inhibited DNA synthesis by regulating the S/G2 transition. The TBBQ concentration giving 50% inhibition in RAW 264.7 cells was 10.71?μg/mL. This threshold value is lower than the residual level of TBBQ in oil, indicating the necessity for concerns over the safety of fried food in terms of TBBQ residues.