Patients with acute lymphoblastic leukemia (ALL), who develop antiasparaginase antibodies without clinical allergic reactions (“silent inactivation”) during l-asparaginase (l-Asp) treatment, have poor outcomes. Ammonia is produced by hydrolysis of asparagine by l-Asp. We postulated that plasma ammonia level might reflect the biological activity of l-Asp. Five children with ALL treated according to the Tokyo Children’s Cancer Study Group (TCCSG) protocol were enrolled. Plasma ammonia levels were analyzed immediately and 1 h after incubation at room temperature and “ex vivo ammonia production” was defined as increase in ammonia concentration. Ex vivo ammonia production well correlated with l-Asp activity (r = 0.882, P < 0.01, n = 23). It always exceeded 170 μg/dL (170–345 μg/dL) in induction therapy. We found 3 patients whose ammonia production was negligible during later phases of therapy. Antiasparaginase antibody was detected and l-Asp activity decreased in these patients. Ex vivo ammonia production is a surrogate marker of l-Asp biological activity.