One possible source of bioactive proteins has been identified: barley flour protein (BFP). An extraction process was optimised through the use of a Box-Behnken Design (BBD). The results showed that an extraction temperature of 54.5ᵒC, a buffer-to-sample ratio of 41.8 mL/g, as well an extraction period of 120 minutes were the ideal parameters for achieving the greatest protein yield (17.8%). For these settings, the computed desirability value was 0.47 %. The BFP electrophoresis analysis showed that two different protein types were present. One kind was made up of low molecular weight proteins, known as globulins of the (2S) type, that ranged in size from 6 to 98 kDa. Furthermore, the presence of lectins was confirmed by a noticeable protein band of 27.6 kDa. Furthermore, in lipopolysaccharide (LPS)-induced RAW264.7 macrophages, the proteins isolated under optimal circumstances showed action in suppressing the release of nitric oxide (NO) and pro-inflammatory inducers such IL-6, IL-1β, and TNF-α. These results imply that BFP may be used as a useful nutraceutical, offering proteins that are both functional and health-promoting.