The heterogeneity and complexity of nonlymphoid tissues has become a major obstacle for the study of immune populations. For this reason, the generation of highly reproducible protocols that allow the analysis of immune cells in these tissues has become crucial for clinical and preclinical research. Here we describe an optimized method that allows the obtention of single-cell suspensions from the skin and lungs to analyze and quantify populations of tissue-resident memory CD8+ T cells by multi-parametric flow cytometry.