Leukemia is a type of blood cancer where abnormal and immature leucocytes are produced in the bone marrow. Methadone hydrochloride is a man-made drug that is commonly used in the maintenance treatment for drug addiction. The objective of this research was to determine the cytotoxic activity and apoptotic effects of methadone hydrochloride treatment towards two leukemia cell lines which are CCRF-CEM and HL-60. CCRF-CEM and HL-60 cells were treated with methadone hydrochloride for 24 and 48 hours to determine the cytotoxic activity. IC50 at 24 hours obtained for CCRF-CEM was 121.6μmol/L while IC50 for HL-60 cells was 97.18μmol/L. Result obtained from DNA fragmentation assay showed no characteristic DNA ladder pattern in CCRF-CEM leukemia cells treated with methadone hydrochloride. Characteristics DNA ladder pattern was observed in methadone hydrochloride treated HL-60 cells. Formation of comets was seen in methadone hydrochloride treated CCRF-CEM and HL-60 cells with varying degree of DNA damage. The comets formed by methadone hydrochloride treated HL-60 cells were more prominent as compared to methadone-treated CCRF-CEM cells. The expression of apoptotic-related proteins in methadone-treated CCRF-CEM and HL-60 cells were checked by incubating the cell lysate with Raybio® Human Apoptosis Antibody Array. Significant alterations in expression level of apoptosis-related proteins in methadone hydrochloride treated CCRF-CEM cells were found involving upregulation of caspase-8 expression and downregulation of survivin expression. Methadone hydrochloride induced apoptosis in HL-60 cells involved upregulation of Bid and caspase-8 expression and downregulation of Bcl-2, p21 and survivin expression.