This article provides a detailed protocol for ion mobility–mass spectrometry (IM-MS) analysis of large protein assemblies. The protocol covers data collection, interpretation, and modeling strategies. Key aspects include optimizing IM separation conditions, calibrating drift times to collision cross-sections, and using molecular modeling to analyze data. The authors discuss the limitations of IM-MS for protein complex analysis, such as the difficulty in ionizing hydrophobic membrane-bound proteins and the need for higher IM resolution for very large complexes. The protocol is designed to help researchers accurately determine the structure of complex protein assemblies, which is crucial for understanding their functional roles.This article provides a detailed protocol for ion mobility–mass spectrometry (IM-MS) analysis of large protein assemblies. The protocol covers data collection, interpretation, and modeling strategies. Key aspects include optimizing IM separation conditions, calibrating drift times to collision cross-sections, and using molecular modeling to analyze data. The authors discuss the limitations of IM-MS for protein complex analysis, such as the difficulty in ionizing hydrophobic membrane-bound proteins and the need for higher IM resolution for very large complexes. The protocol is designed to help researchers accurately determine the structure of complex protein assemblies, which is crucial for understanding their functional roles.