Immuno-oncology (IO) research demands robust tools to characterize immune responses in solid tumors and peripheral blood. High-dimensional flow cytometry enables simultaneous assessment of lineage, activation, exhaustion and functional markers, providing critical insights into mechanisms of response and resistance to immunotherapies. Precision for Medicine developed and validated a 32-parameter flow cytometry assay to advance translational research and biomarker discovery in IO clinical trials. This high-parameter panel profiles major immune subsets in cryopreserved PBMCs - including T cells, memory T cell subsets, regulatory T cells, B cells, NK cells and myeloid populations - while assessing activation (CD25, CD69, CD86), exhaustion (PD-1, LAG-3, TIM-3), proliferation (Ki67), and functional markers (Granzyme B, TCF-1), alongside cytokine expression (IL-2, IL-4, IFNgamma, TNFalpha). Validation followed CLSI guidelines for precision, accuracy, and sensitivity using cryopreserved PBMCs from healthy donors. The assay demonstrated reproducibility (CV less than 15% for major immune populations), sensitivity for low-frequency subsets such as TCF-1+ stem-like T cells, and robust functional readouts post-stimulation. Clinical relevance was confirmed through profiling of exhaustion and activation states critical for IO trials and solid tumor studies. This validated high-parameter flow cytometry assay enables scalable immune monitoring in IO clinical trials, supporting biomarker discovery, mechanism of action studies and pharmacodynamic endpoint assessment longitudinally in patients receiving novel immunotherapies. Its reproducibility and functional depth make it suitable for multi-site implementation and integration with computational pipelines, facilitating patient stratification and advanced biomarker insights for solid tumor research.