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The aim of the present study was to address the effect of high hydrostatic pressure (HHP) on bioactive compounds and antioxidant activity of Nelumbo nucifera (Lotus) bee pollen (LBP). The total phenolic contents (TPC) and free amino acids were determined and flavonoid composition was identified by UPLC-Triple-TOF-MS. Five analytical methods including DPPH, ABTS, reducing power, FRAP, and silver nanoparticles antioxidant capacity were used for the antioxidant activity analysis. The results revealed a significant increase in the TPC and free amino acid compositions after HHP treatment. The DPPH, ABTS, reducing power, FRAP, and AgNPAC of the extracted fractions were augmented by over 13.94, 2.9, 2.68, 85, and 2.6 times, respectively. PCA analysis showed a strong correlation between TPC, some distinct flavonoids, and antioxidant activities. These results indicated that HHP enhances the antioxidant activity of LBP via increase in amino acids, TPC, and some flavonoids such as Licoagrone, Kuwanon K, Cyanidin 3-rutinoside, etc.