In this study, we evaluated the impact of gamma irradiation, heat inactivation and neutral buffered formalin fixation on phospholipid profiles in mouse kidney tissue using MALDI-MSI. By combining class-level normalization to deuterated standards, relative percentage change analysis, and receiver operating characteristic (ROC)–based species discrimination, we provide a quantitative framework to assess the robustness and sensitivity of lipid measurements across commonly used tissue inactivation methods. This dataset is the negative ion mode SCiLS assembly exported in the imZML format. Please write to the authors to request raw or supporting data due to data volume. Reasonable requests will be granted.