Processing and Analyzing High-Throughput Microfluidic Enzymology Data: A Practical Guide To Rate Fitting and Quality Control

High-throughput enzymology enables quantitative characterization of enzyme function across hundreds to thousands of sequence variants and experimental conditions. Nevertheless, the scale and complexity of these datasets create substantial challenges for analysis and quality control. High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), for example, generates large microscopy datasets that must pass through multiple analytical stages, including image processing, initial-rate fitting, and kinetic modeling. Choices or errors made at any stage can propagate into the final kinetic parameters without being evident from fit statistics alone. Here, we provide a broadly applicable guide for analyzing high-throughput enzymology data from the HT-MEK platform, using Michaelis-Menten kinetics as a representative example. We first outline the conceptual workflow from raw fluorescence measurements to estimates of kcat, KM, and kcat/KM. We then discuss common experimental and analytical failure modes and present a framework for deciding when data should be refitted, filtered, qualified, or repeated. Finally, we provide a step-by-step workflow using Mercury, an open-source Python framework that integrates scalable HT-MEK data processing with traceable quality control and diagnostic visualization. This workflow preserves the connection between reported parameters and their underlying measurements and can be adapted to other kinetic models and biochemical assays.