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Context The underground edible tuber of Dioscorea alata L. (Dioscoreaceae) is a functional food with high nutritive value and therapeutic potential. The tuber is known to possess anti-inflammatory properties in traditional medicine. Objective The present study explores the anti-inflammatory activity and standardisation of D. alata tuber hydromethanol extract. Materials and methods Hydromethanol extract (70%) of D. alata tuber was chemically characterised using HPLC and GC-MS techniques. Murine lymphocytes were cultured for 48 h with six different concentrations (0-80 μg/mL) of the extract. The expression of nitric oxide (NO), TNF-α, COX-1, COX-2, and PGE2 were evaluated using colorimetric and ELISA methods. Results Dioscorea alata extract inhibited the expression of NO and TNF-α with an IC50 value of 134.51 ± 6.75 and 113.30 ± 7.44 μg/mL, respectively. The IC50 values for inhibition of total COX, COX-1, COX-2 activities and PGE2 level were 41.96 ± 3.07, 141.41 ± 8.99, 32.50 ± 1.69, and 186.34 ± 15.36 μg/mL, respectively. Inhibition of PGE2 level and COX-2 activity was positively correlated (R(2) = 0.9393). Gallic acid (GA), 4-hydroxy benzoic acid (4HBA), syringic acid (SYA), p-coumaric acid (PCA), and myricetin (MY) were identified and quantified using HPLC. GC-MS analysis revealed the presence of 13 different phytocompounds such as hexadecanoic acid, methyl stearate, cinnamyl cinnamate, and squalene. Conclusion The D. alata extract significantly down-regulated the pro-inflammatory signals in a gradual manner compared with control (0 μg/mL). Different bioactive phytocompounds individually possessing anti-inflammatory activities contributed to the overall bioactivity of the D. alata tuber extract.