Background: L-asparaginase (ASNase) is a key drug in the treatment of ALL. ASNase therapy aims to lower serum asparagine (ASN) levels, but no critical minimum value for efficacy has yet been established. ASN levels are difficult to measure accurately due to ex vivo depletion during the time required to harvest plasma from blood samples and to quench the enzyme, even if samples are immediately processed and stored on ice. Eryaspase is an investigational product under development. Following infusion of eryaspase, ASN is actively transported into RBCs, where it is hydrolyzed by the encapsulated ASNase. Methods: This randomized Phase 2/3 study enrolled pts with relapsed ALL. The co-primary endpoints were the mean duration of ASNase activity > 100 U/L and incidence of allergic reactions during the induction phase. Secondary endpoints were safety, complete remission (CR), pharmacokinetics (PK), and pharmacodynamics (PD). Pts (n = 80, age:1-55 years) were randomized to eryaspase or native ASNase. Results: The mean duration of ASNase activity > 100 U/L measured in whole blood was significantly higher with eryaspase (18.9 ± 5.3 days) compared with native ASNase (8.5 ± 6.6 days). In both treatment arms, ASN depletion ≤2 µM was maintained for ~7 days in 75% of pts. The mean duration of ASN depletion ≤2 µM was 6.0 ± 5.0 and 11.6 ± 7.3 days with eryaspase and native ASNase, respectively. Exploratory receiver operating characteristic (ROC) analysis suggested that an optimal threshold of ≤ 7.55 µM ASN on Day 6 correlated with CR with a positive predictive value of 0.88. Conclusions: The assumed requirement for prolonged ASN depletion in patients receiving ASNase therapy is likely to be an overestimation caused by ex-vivo depletion that is observed with free ASNase. Measurement of ASN depletion in pts treated with eryaspase are less prone to such error. Accordingly, the efficacy of encapsulated ASNase cannot be accurately compared with that of free ASNase based on ASN depletion. ASN depletion ≤2 μM may not be needed with eryaspase, and a level ≤7.55 µM correlated with CR. Clinical trial information: NCT01518517