In this protocol, we present Epi-CyTOF, a cytometry by time of flight (CyTOF) approach for single-cell epigenome profiling in cortical organoids as a model system for human brain development. We describe steps for in silico panel design, antibody conjugation and testing, sample preparation and staining, and high-dimensional data analysis. Additionally, we provide recommendations for applying Epi-CyTOF to other model systems based on our experience with gastroesophageal tumoroids. For complete details on the use and execution of this protocol, please refer to Ditzer et al .1.