This book constitutes the refereed proceedings of the 16th International Conference on Computational Methods in Systems Biology, CMSB 2018, held in BRNO, Czech Republic, in September 2018. The 15 full and 7 short papers presented together with 5 invited talks were carefully reviewed and selected from 46 submissions. Topics of interest include formalisms for modeling  biological processes; models and their biological applications; frameworks for model verification, validation, analysis, and simulation of biological systems; high-performance computational systems biology; parameter and model inference from experimental data; automated parameter and model synthesis; model integration and biological databases; multi-scale modeling and analysis methods; design, analysis, and verification methods for synthetic biology; methods for biomolecular computing and engineered molecular devices. Chapters 3, 9 and 10 are available open access under a CreativeCommons Attribution 4.0 International License via link.springer.com.