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Our understanding of programmed cell death 1 (PD‐1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD‐1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD‐1 ligation on short‐term T cell signaling, long‐term T cell function, and the structural consequences of PD‐1 ligation with PD‐1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short‐term assay to examine the signaling cascade triggered by PD‐1. We describe a phospho flow cytometry method to determine how PD‐1 ligation alters the level of CD3ζ phosphorylation on Tyr¹⁴², which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate‐bound assay that is useful to examine the long‐term consequences of PD‐1 ligation such as cytokine production and T cell proliferation …