Peripheral blood mononuclear cells (PBMCs) are essential to the study of autoimmune, infectious, parasitic diseases, and cancer. In the rapidly growing field of cancer immunology, cellular phenotyping provides critical information about patient responses to treatments and treatment efficacies. Notably, the evaluation of T cell based therapies relies on the isolation of highly viable CD3+ T cell, CD4+ Helper T cell, and CD8+ Cytotoxic T cell populations before and during patient treatments. Cryopreservation of PBMC populations allows researchers to thaw and characterize clinical samples by flow cytometry, mass cytometry, sequencing, etc. in a high-throughput manner and in batches. Therefore, it is important to separate and bank an abundance of robust circulating immune cells. Here, we report our internal protocols for the high-quality separation, banking, and thawing of clinically relevant PBMC populations. We present quality control data from 11 melanoma patients and characterize their CD3+, CD4+, and CD8+ T cells by 4-color flow cytometry.