Enzymes provide attractive targets for drug development. Quantitative analysis of enzyme reactions can lead to rational drug discovery. For undergraduates majoring in biochemistry or pharmacy and engaged in drug discovery-related work, it is essential to acquire a comprehensive understanding of enzyme kinetics, including experimental methods and the fundamental principles of drug screening. Being overexpressed in cancer, MTHFD2 is an emerging anticancer drug target. Herein, we used MTHFD2 to develop a high-throughput drug screening laboratory course based on enzyme kinetics for upper-division undergraduate students. The learning activities that were designed for students included "Escherichia coli"-based protein expression, protein purification, enzyme activity assay, setting up a high-throughput drug screening assay, and data analysis. The main purpose of this one-week laboratory education course is to provide students with a better understanding of enzyme kinetics, high-throughput drug screening and to help them grasp the relationship between enzyme kinetics and drug discovery. This hands-on course provides students an opportunity to learn how theoretical knowledge is applied in real practice.