L-asparaginase (L-ASNase) is an essential component of chemotherapy schemes due to its differential action over normal and leukemic cells. Recently, concerns on the performance of commercial formulations administered in developing countries have been reported. To address this problem, we developed a cell-based protocol to compare the activity of different L-ASNase formulations used in Colombia. We found three statistically different groups, ranging from full to no activity on leukemic cells using 0.05, 0.5 and 5.0 IU/mL concentrations. According to our results, we advise a preclinical evaluation for formulations of L-ASNase distributed in developing countries which could impact the outcome of patients.