Suppressing the aversive bitterness of bioactive peptides is an arduous task as it hinders product acceptability. Three acquisition modes (ddMS, vDIA, and mDIA) of high-resolution mass spectrometry (HRMS) were designed for structure confirmation and accurate quantification of HPFLEWAR, with the mDIA mode chosen as optimum. HRMS and isothermal titration calorimetry was used to elucidate the mechanism that β-lactoglobulin self-assemble to form association complex in 1:1 stoichiometric ratio (ΔG value - 29.36 kJ mol), which automatically attracted HPFLEWAR and reduces its distribution in free form, downgraded the level of bitter perception. Proteomics experiments and molecular dynamics simulations was built to discovered that HPFLEWAR bound and stabilized in the negatively charged region of β-lactoglobulin via four hydrogen bonds (Lys69, Ile72, Asp53, and Glu74) and hydrophobic interactions. These findings were considered to give theoretical foundation for strictly controlling the bitter perception of peptides and the possible application of HPFLEWAR as new functional components.