Updated project metadata. Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain essential as organoid models evolve. To solve this problem, we have designed a workflow to reproducibly extract proteins from brain organoids, measure global protein turnover using mass spectrometry, and statistically investigate turnover differences between genotypes. We also provide robust methodology for data filtering in turnover determination and for statistical treatment of turnover data. Using human midbrain organoids (hMO) as a model system, our method accurately characterized the half-lives of 773 midbrain proteins. We compared these half-lives both to Parkin knockout hMOs and to previously reported data from primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.