Tumor Necrosis Factor Alpha (TNF-α) is a pleiotropic pro-inflammatory cytokine. It act as central biological regulator in critical immune functions, but its dysregulation has been linked with a number of diseases. Inhibition of TNF-α has considerable therapeutic potential for diseases such as cancer, diabetes, and especially autoimmune diseases. Despite the fact that many small molecule inhibitors have been identified against TNF-α, no orally active drug has been reported yet which demand an urgent need of a small molecule drug against TNF-α. This study focuses on the development of ligand-based selective pharmacophore model to perform virtual screening of plant origin natural product database for the identification of potential inhibitors against TNF-α. The resultant hits, identified as actives were evaluated by molecular docking studies to get insight into their potential binding interaction with the target protein. Based on pharmacophore matching, interacting residues, docking score, more affinity towards TNF-α with diverse scaffolds five compounds were selected for in vitro activity study. Experimental validation led to the identification of three chemically diverse potential compounds with the IC50 32.5 ± 4.5 µM, 6.5 ± 0.8 µM and 27.4 ± 1.7 µM, respectively.