Peptides with a similar antioxidant and ACE-inhibitory activity of royal jelly (RJ) generated from Alcalase hydrolysated pollen (AHP) were predicted by Response Surface Methodology (RSM). The model equations were proposed according to the effects of time and enzyme concentration on the antioxidant and ACE-inhibitory activity. The optimum values for Alcalase concentration and hydrolysis time were 1.5% and 4 h, respectively. Later, AHP was prepared and deproteinised to be further analysed using size-exclusion chromatography (SEC). After SEC separation, fractions with the highest activity of ACE-inhibitory, DPPH radical scavenging and ferric-reducing power were purified by RP-HPLC. The highest ACE-inhibitory and DPPH scavenging activity of fractions was found 100% and 66.61%, respectively. The most active fractions were analysed by nano-liquid chromatography and mass spectrometry in tandem (nLC-MS/MS) and a total of 195 peptide sequences were identified. The origins of all peptides were herbal proteins and certain coincidences with previously described bioactive sequences were discussed.