Asparaginase (ASNase) is one of the first‐lines of chemotherapy used in the treatment of acute lymphoblastic leukemia (ALL). It breaks down asparagine in the body forcing the cell to undergo apoptosis. Yet, some patients develop resistance to ASNase treatment. Asparagine is produced in the cell by the enzyme asparagine synthetase (ASNS). One explanation for resistance could be increased ASNS expression leading to the production of more asparagine than can be broken down. Here, we investigated whether using a competitive ASNS inhibitor, albizziine (Alb), to decrease ASNS activity would reverse ASNase resistance in leukemic cell lines. Two human leukemic cell lines with varying ASNS expression were utilized, Jurkat and HL‐60. Real time‐PCR analysis showed an 8.9‐fold higher level of ASNS mRNA in HL‐60 relative to Jurkat. In addition, that HL‐60 was resistant and Jurkat sensitive to ASNase‐induced apoptosis; as determined by staining the cells with Annexin V‐FITC and flow cytometric analysis. After 24 hours, 0.1 IU ASNase treatment resulted in a 43.27% increase in apoptosis in Jurkat cells, but no significant increase in HL‐60 cells. The addition of the ASNS inhibitor, Alb, in combination with ASNase resulted in increased apoptosis in both cell lines. HL‐60 apoptosis increased 6‐fold when treated with a combination of 0.1 IU ASNase and 1mM Alb. Alb also increased Jurkat sensitivity to 0.1 IU ASNase from 43.27% apoptosis with ASNase alone to 67.15% in combination with ASNase. Alb alone did not induce any significant death. These results suggest that the ASNS inhibitor, Alb, can restore sensitivity to ASNase induced death in leukemic cell lines and therefore warrants further investigation into its role in reversing ASNase chemotherapeutic resistance.Support or Funding InformationThis work was supported by the Honors Program at Elon University, Elon Undergraduate Research Program, and Glen Raven grant.