CD28 is a critical costimulatory receptor involved in T cell activation and immune regulation, making it a compelling target for immunomodulatory therapies. Despite its therapeutic relevance, small molecule CD28 inhibitors remain largely underexplored. To address this gap, we developed a high-throughput screening (HTS) workflow using surface plasmon resonance (SPR) to identify novel CD28-targeted small molecules. To our knowledge, this work represents the first SPR-based HTS platform applied to the discovery of small molecules targeting a stimulatory immune checkpoint receptor. A chemical library composed of diverse 1056 small molecules was screened using a 384-well format. Compounds were evaluated based on level of occupancy (LO), binding response, and dissociation kinetics, resulting in 12 primary hits (1.14% hit rate). Follow-up dose-response SPR screening confirmed micromolar-range affinities for three compounds. Molecular docking and 100 ns molecular dynamics simulations of the top hit, DDS5, revealed a stable complex with CD28, maintained by hydrogen bonding and a persistent interaction with Phe93. Functional validation using a competitive ELISA confirmed that DDS5 inhibited the CD28-CD80 interaction. These results demonstrate that our SPR-based HTS platform is a robust and efficient strategy for discovering CD28-targeted small molecules. The integration of computational evaluation and orthogonal validation further underscores the potential of DDS5 as an early stage immunomodulatory agent.