l-asparaginase is an enzyme produced by microorganisms, plants, and animals, which is used clinically for the treatment for acute lymphoblastic leukemia (ALL) and, in the food industry, to control acrylamide formation in baked foods. The purpose of this review was to evaluate the available literature regarding microbial sources of l-asparaginase, culture media used to achieve maximum enzyme expression in microbial fermentations, and assay methods employed to assess l-asparaginase activity. Studies were gathered by searching PubMed, and Web of Science databases before January 22, 2018, with no time restrictions. The articles were evaluated according to the source of l-asparaginase being studied, the nitrogen source in the culture medium, the type of sample, and the method employed to evaluate l-asparaginase activity. Bacterial l-asparaginase appeared to be the most commonly studied source of the enzyme and, most often, the enzyme activity was assayed from crude protein extracts using the Nessler method, which is an indirect measurement of asparaginase activity that determines the concentration of ammonia generated after the action of the enzyme on the substrate, l-asparagine. However, ammonia is also generated throughout microbial fermentations and this endogenous ammonia will also reduce the Nessler reagent if crude microbial extracts are used to determine total l-asparaginase activity. We suggest that current estimates of l-asparaginase activity reported in the literature may be overestimated when Nessler reagent is used, since we were unable to find a single study that made reference to the possible inference of fermentation derived ammonia.