Background: Asparaginase is one of the essential chemotherapies used to treat acute lymphoblastic leukemia (ALL). Asparaginase antibody production may cause a subtherapeutic level and result in an inferior outcome. Methods: Prospective study of asparaginase activity among patients who received native E.coli asparaginase 10,000 IU/m2 according to ThaiPOG ALL protocol. The asparaginase activity is measured by the coupled enzymatic reaction. Pharmacokinetic data includeding peak activity (Cmax), time to maximum concentration (Tmax), area under the curve (AUC0-48h) being elucidated. Results: Eight patients (five males and three females), median age 9.5 years, were enrolled. The median asparaginase activity of seven cases who are eligible for calculation reached Tmax within 24 hours (range 6-48 hours) with mean Cmax 3.60±0.34 (range 3.02-4.11) IU/ml. Mean AUC0-48h is 143.23±36.94 IU.h/mL (range 71.07 – 180.12 IU.h/mL). The post-48-hour activity showed a mean of 3.19±0.24 IU/ml (range 2.77-3.51 IU/ml) which implied an adequacy of activity over 48 hours and proper for the 12-day period. One relapsed ALL patient showed an extremely low AUC of asparaginase activity which coincided with urticaria after asparaginase injection. Subsequently, the asparaginase antibody was demonstrated in this patient. Conclusion: Native E. coli asparaginase-based protocol provides a compelling pharmacokinetic effect. Plasma asparaginase activity and antibody testing are recommended for a relapsed patient or a suspected allergic reaction to the asparaginase. Patients with low asparaginase activity may benefit from switching to an alternative form of asparaginase to maintain treatment efficacy.