In 2016, the National Science Foundation (NSF) established a five-year program on Understanding the Rules of Life (URoL) to identify generalizable rules that govern biological systems at micro and macro levels. The National Academies of Sciences, Engineering, and Medicine has been requested by NSF to conduct a retrospective analysis of the URoL program. This analysis involves understanding the program’s: a) achievements; b) integration of multi-disciplinary, systems-level approaches; c) broader implications for studying highly-complex systems, future scientific questions, and future societal needs; and d) the production of generalizable rules that apply to different fields and scales.