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This study was designed within the scope of industrial exploitation of antioxidant extracts from Hibiscus sabdariffa. Efficiency in the extraction of antioxidants (total phenolics, flavonoids and anthocyanins) from calyces was assessed through different processes involving solvents (water, 80% acetone and 80% methanol), microwaves and ultrasounds. Results indicated that microwave extraction with 80% acetone gave the highest yield for total phenolics, expressed as gallic acid equivalents (GAE) per g dry weight (DW) (3.73 GAE/100 g DW), while the highest results for total flavonoids and anthocyanins were found in the microwaved aqueous extracts. Antioxidant activity of the extracts reported the highest reducing power for aqueous extracts with microwave extraction, while 80% acetone and methanol samples exhibited the highest antioxidant activity by high-performance thin-layer chromatography (HPTLC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. HPTLC performed for screening of phenolic compounds revealed the occurrence of anthocyanins in dimer-like bands. Laboratory tests showed high protection against lipid oxidation of the whey spread emulsion (22.32 h) incorporated with the sonicated aqueous extract. Results of peptide sequencing data by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, revealed protein–polyphenols interactions by the formation of dimers between polyphenols and protein/peptides. These interactions were found without effect on the oxidative stability of the emulsions. These study suggest that H. sabdariffa’s calyces have the potential to be used like food antioxidants.