This book constitutes the refereed proceedings of the 21st International Conference on Computational Methods in Systems Biology, CMSB 2023, held in Luxembourg City, Luxembourg, during September 13–15, 2023.The 14 full papers and 3 tool papers presented in this book were carefully reviewed and selected from 28 submissions. CMSB focuses on modeling, simulation, analysis, design and control of biological systems and covers the broad field of computational methods and tools in systems and synthetic biology and their applications.