Background L‐asparaginase is a cornerstone treatment for children with acute lymphoblastic leukemia (ALL). However, immune reaction to the drug may increase the clearance or impair the function of L‐asparaginase and reduces its therapeutic efficacy. The objective of this study was to identify potential plasma proteins that could be used as proxies for L‐asparaginase activity. Methods Fibrinogen, von Willebrand factor antigen (VWF:Ag), total protein, and albumin levels as well as antithrombin (AT) and L‐asparaginase activities were measured in 97 children with ALL treated for prolonged period of time with L‐asparaginase. Binary logistic regression and a receiver operating characteristic (ROC) curve analysis were performed to evaluate the predictive value of plasma proteins for L‐asparaginase activity. Results Median E. coli L‐asparaginase activity was 220 IU/L (range, 0–1308) throughout the treatment period. L‐asparaginase activity was below 100 IU/L in 23% of measured samples. L‐asparaginase activity was inversely associated with AT activity, fibrinogen, total protein, and albumin levels (r = −0.63, −0.62, −0.57, and −0.45, respectively; P < 0.0001), but not with VWF:Ag. ROC curve analyses showed an intermediate accuracy of AT activity (area under the ROC curve [AUC] = 0.77) to detect specimens with subtherapeutic level of L‐asparaginase. An optimal accuracy was found when AT and fibrinogen were combined (AUC = 0.82; sensitivity = 75%; specificity = 82%; positive predictive value = 55%; negative predictive value = 92%) with cutoff values of 0.73 IU/mL and 1.85 g/L, respectively. Conclusions AT combined with fibrinogen levels could be used as a proxy to identify patients with therapeutic level of L‐asparaginase activity in the absence of real‐time asparaginase measurement during prolonged exposure to L‐asparaginase.