Background: Diabetes is becoming a serious threat to human health worldwide and has affected quality of life in Taiwan. Acetylsalicylic acid (ASA) is a commonly used drug for preventing cardiovascular disease in patients with type 2 diabetes mellitus in Taiwan. However, the effect of ASA on the reduction of the defection of beta cells remains unclear. Method: The protective effects of ASA on the INS-1 cell line was evaluated following the exposure of the cells to normal glucose level (11 mM) or high glucose level (33 mM) to mimic normoglycemia and hyperglycemia situations, respectively. Possible action mechanisms were determined by analyzing the generation of reactive oxygen species (ROS), nitrites, and lactate dehydrogenase (LDH). Results: Although the effect of ASA was not significant in the normal glucose condition, ASA significantly reversed the elevation of ROS, nitrites production, and LDH levels induced by high glucose in INS-1 cells. Conclusions: ASA results in anti-cell damage under hyperglycemic conditions through a decrease in LDH levels. However, additional studies using diabetes models are required to validate this finding and investigate the underlying mechanism. Furthermore, the manipulation of intracellular LDH levels in β-cells are a potential therapeutic focus for hyperglycemia. Moreover, LDH level is a potential marker of β-cell damage.