This protocol describes a detailed method for data collection and interpretation in ion mobility–mass spectrometry (IM-MS) analysis of large protein assemblies. Ion mobility separates gaseous ions based on size and shape, and this protocol covers data interpretation, prediction of separation of model structures, normalization, and modeling strategies. It also includes instrument settings and best practices for detecting large noncovalent protein complexes. The introduction discusses the complexity of protein interactions and the challenges in determining their structures. IM-MS is a recent addition to biophysical tools that can determine mass and subunit composition of protein complexes. The protocol outlines the process of ionizing the protein complex, injecting ions into a gas-filled region, and separating them based on ion-neutral collision cross-section. After separation, ions are analyzed by mass spectrometry based on their mass-to-charge ratio. The protocol addresses the feasibility of separating likely protein complex structures, the general features of IM-MS data, instrument conditions for optimal separation and mass measurement, and the conversion of drift time data to collision cross-section. It also discusses molecular modeling for data analysis, limitations of IM-MS, and the calibration of traveling-wave IM drift times. The protocol includes steps for generating and analyzing data, troubleshooting common issues, and interpreting results. It emphasizes the importance of calibration, the influence of ion charge and mass on resolution, and the use of molecular modeling to generate and compare model structures with experimental data. The protocol also highlights the importance of error estimation and the use of molecular dynamics and docking calculations to generate potential structures. The anticipated results show the effectiveness of IM-MS in analyzing protein complexes, including the ability to remove chemical noise and perform structural studies. The protocol provides a comprehensive guide for researchers to apply IM-MS in the analysis of large protein assemblies.This protocol describes a detailed method for data collection and interpretation in ion mobility–mass spectrometry (IM-MS) analysis of large protein assemblies. Ion mobility separates gaseous ions based on size and shape, and this protocol covers data interpretation, prediction of separation of model structures, normalization, and modeling strategies. It also includes instrument settings and best practices for detecting large noncovalent protein complexes. The introduction discusses the complexity of protein interactions and the challenges in determining their structures. IM-MS is a recent addition to biophysical tools that can determine mass and subunit composition of protein complexes. The protocol outlines the process of ionizing the protein complex, injecting ions into a gas-filled region, and separating them based on ion-neutral collision cross-section. After separation, ions are analyzed by mass spectrometry based on their mass-to-charge ratio. The protocol addresses the feasibility of separating likely protein complex structures, the general features of IM-MS data, instrument conditions for optimal separation and mass measurement, and the conversion of drift time data to collision cross-section. It also discusses molecular modeling for data analysis, limitations of IM-MS, and the calibration of traveling-wave IM drift times. The protocol includes steps for generating and analyzing data, troubleshooting common issues, and interpreting results. It emphasizes the importance of calibration, the influence of ion charge and mass on resolution, and the use of molecular modeling to generate and compare model structures with experimental data. The protocol also highlights the importance of error estimation and the use of molecular dynamics and docking calculations to generate potential structures. The anticipated results show the effectiveness of IM-MS in analyzing protein complexes, including the ability to remove chemical noise and perform structural studies. The protocol provides a comprehensive guide for researchers to apply IM-MS in the analysis of large protein assemblies.