Abstract Background Enzymatic assays are among the most common diagnostic tests performed in the clinical laboratory. Enzymatic substrate analysis is most commonly measured using endpoint methods; however, modulating the reaction kinetics allows fine control of the reaction rate, which can be adjusted based on specific monitoring technologies. Methods We developed and optimized an enzymatic method for measurement of creatinine in plasma, using commonly paired enzymes of creatininase (Crtnnase), creatinase (Crtase), sarcosine oxidase (SOX), ascorbate oxidase (AOX), and horseradish peroxidase (HRP). The novel aspect of the assay is that it is fast and uses SOX as the limiting enzyme. The assay performance was assessed with respect to precision, accuracy, and interferences. Results The intrarun %CV (n = 12) was approximately 5% for each concentration tested, with biases ranging from −3 to −9%. The interrun %CV (n = 39) ranged from 5 to 8%, with biases ranging from −2 to −6%. During the accuracy assessment (n = 127), only 4 samples did not meet the minimum acceptability criteria. Minimal interference was observed, except at low creatinine concentrations with elevated creatine. Conclusion Our novel and versatile enzymatic assay to measure plasma creatinine using kinetic analysis with SOX as the limiting enzyme is rapid (<2 mins), sensitive, and specific and demonstrates excellent concordance with the laboratory standard. We anticipate this rapid kinetic assay to be compatible with emerging technologies in the field of portable diagnostic devices, such as the usage of silicon photonics to monitor biochemical reactions.