PD‐1/PD‐L1 is a critical druggable target for immunotherapy against sepsis. Chemoinformatics techniques involved the structure‐based 3D pharmacophore model development followed by virtual screening of small molecule databases to identify the small molecules against PD‐L1 pathway inhibition. Raltitrexed and Safinamide act as potent repurposed drugs, and three other Specs database compounds using in silico methods. These compounds were screened based on the pharmacophore fit score and binding affinity towards the active site of the PD‐L1 protein. In silico pharmacokinetic profiling of these screened compounds was done to test their biological activity. Next, experimental validation of the best four virtually screened hits was done in vitro for its hemocompatibility and cytotoxicity. Among these, Raltitrexed, Safinamide and Specs compound (AK‐968/40642641) effectively increased the proliferation of immune cells and IFN‐γ production. These compounds can act as potent PDL‐1 inhibitors for adjuvant therapy against sepsis.