Background: Inter-K Peptide Therapeutics has developed two synthetic compounds that modulate kinase activity. To investigate the potential of these compounds as cancer immunotherapeutics, a flow cytometric intracellular cytokine staining (ICS) assay was developed to investigate the effect of the compounds on immune cells isolated from tumors from subjects with non-small cell lung cancer (NSCLC). In addition, the concentration of various cytokines was measured in cell culture supernatant after treatment with the compounds. Methods: After optimizing the assays in PBMC from healthy donors, the assay was used for clinical specimens. Tumor infiltrating lymphocytes (TILs) were isolated from two resected tumors by mechanical disaggregation. TILs were stimulated with or without anti-CD3/CD28 antibodies with and without Inter-K Compound A or Compound B for 72 hours. Following stimulation, cell culture supernatants were analyzed for 10 proinflammatory-related cytokines using the Meso Scale Discovery (MSD) assay. TILs were analyzed by flow cytometry with a 10-color immunophenotyping panel to measure the expression of functional markers CD25, IL-2, IFNg and Ki67 in NK, NKT, CD4 T and CD8 T cells. Results: Immunomodulation induced by Compound A treatment included an over two-fold increase in CD25 expression in CD4 T cells, statistically significant increases in IL-6 and IL-8 production while a decrease in Ki67 expression in CD8 T cells and a reduction in IL-2, IFNg, TNFa, IL-4, IL-10 and IL-13 production was observed. Compound B did not appear to have immunomodulatory properties. Conclusions: This work provides an example of the utility of flow cytometry in compound evaluation. In this case, preliminary data suggests that Compound A but not B was able to modulate the response in TIL samples in vitro, and supports the continued investigation of Compound A as an immunotherapeutic. Although TIL specimens are challenging to work with due to tumor availability and low cellular recovery, as the target for the immunotherapy, it is important that compound characterization be conducted in TIL in addition to healthy donor PBMC and cell lines.