Peripheral blood smear microscopy still remains the gold standard for diagnosing malaria and trypanosomiasis. Microscopy is a labor-intensive process and requires great amount of skill to accomplish. Even though cheap and easy to perform, it still has several limitations. This hinders the microscopist in identifying protozoan structure or differentiating species from one another. Considering these factors in the performance of microscopic examination, it is crucial to identify new strategies for parasite identification and species differentiation. Innovations in clinical enzymology, immunodiagnostics, and molecular technology would be of help in resolving the problem. This study mainly focused on the possible role of clinical enzymes in malaria and trypanosomiasis diagnosis. Enzymes play a vital role in parasite physiology and metabolism. They enable the parasite to survive inside the living host by initiating different metabolic cycles. These enzymes can either be expressed on the surface of the protozoa or excreted in the extracellular environment. Lactate dehydrogenase, aldolase, and glutamate dehydrogenase were the significant enzymes associated with pathogenic Plasmodium spp. Other malarial enzymes were also identified but further validation is still required to establish their use as diagnostic biomarkers. Whereas, the enzyme matrix metalloproteinase was identified as significant for diagnosing and differentiating Trypanosoma spp. Analysis of these enzymes can be used as alternative means for microscopy in parasite identification and differentiation. Application of these enzymes as immunologic markers in various diagnostic test kits should be further evaluated.