L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylatedEscherichia coli(E.coli)-derived ASNase as well asErwinia chrysanthemi-derived ASNase. Additionally, a new recombinantE.coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinantE.coli-derived ASNase marketed in Europe (Spectrila®) with anE.coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila®also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, theE.coliDNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila®met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited.