This book constitutes the refereed proceedings of the 20th International Conference on Computational Methods in Systems Biology, CMSB 2022, held in Bucharest, Romania, in September 2022.The 13 full papers and 4 tool papers were carefully reviewed and selected from 43 submissions. CMSB focuses on modeling, simulation, analysis, design and control of biological systems. The papers are arranged thematically as follows: Chemical reaction networks; Boolean networks; continuous and hybrid models; machine learning; software.