Background: Treatment of acute promyelocytic leukemia (APL) a subtype of acute myeloid leukemia (AML) develops as a result of a series of genetic mutations in a hematopoietic precursor cell. The incapability to undergo apoptosis is a key mechanism of multidrug resistance in AML, and the analysis of autophagy and apoptotic factors may demonstrate a significant prognostic tool to predict the outcome. The pro-survival func-tion of autophagy is known to be adaptive, but in the context of cancer, is significantly abnormal. Resistance cell death owing to activation of autophagy prosurvival in cancer cells. In this study, we evaluate the level of LC3II and caspase3 after treat-ment NB4 cell line by Arsenic trioxide, All-trans retinoic acid, 3-MA, BafA1 and HCQ.Materials and Methods: We studied in vitro effects of Arsenic trioxide, All-trans retinoic acid, 3-MA, BafA1 and HCQ on hu-man leukemia cell line (NB4) in a dose-dependent and time-dependent manner which investigated through cell proliferation assay in both single and combination doses. Next the rate of apoptosis (annexin V FITC assay) and cell cycle arrest meas-ured by flow cytometry. The development of acidic vesicular organelles (AVOs) specified by acridine orange (AO) staining of the cells, then Photos were obtained with a fluorescence mi-croscope. In addition, we evaluate the level of LC3II protein by western blot. And finally, the level caspase3 mRNA expression was evaluated via real-time PCR.Results: The data show that a combination of ATO (1μM) and ATRA (700nM) and HCQ effectively induced apoptosis and decreased leukemic cell proliferation. Real-time PCR analysis indicated that mRNA expression of Cas3 was increased which was accompanied by accumulation of LC3II protein.Conclusion: These in vitro studies imply that ATO/ATRA/HCQ has a direct antileukemic effect by inhibiting autophagy factors.