Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug L-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine-dependency of ALL cells. In addition to hydrolyzing the amino acid L-asparagine, all FDA-approved L-asparaginases also have significant L-glutaminase coactivity. Since several reports suggest that L-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced L-glutaminase coactivity might be clinically beneficial if their anti-leukemic activity would be preserved. Here we show that novel low L-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi L-asparaginase were highly efficacious against both T and B cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer L-asparaginases without L-glutaminase activity for the treatment of human ALL.