The aim of the present study was to report on a complete synthetic approach, namely benzylation-hydrolysis-acylation-hydrogenation, to the synthesis of regioselectively acylated quercetin analogues using low-cost rutin as a starting material. Three quercetin analogues, quercetin-3-O-propionate (Q-pr), quercetin-3-O-butyrate (Q-bu) and quercetin-3-O-valerate (Q-va), containing 3-, 4- and 5-carbon aliphatic acyl chains, respectively, were synthesized and characterized with 1H nuclear magnetic resonance (NMR), 13C NMR and mass spectrometry. Compared with quercetin, all three analogues exhibited improved lipophilicity. The lipophilicity of the analogue increased with increasing acyl chain length. Q-va exhibited the highest lipophilicity among the three analogues, but a lower water solubility compared with quercetin. By contrast, Q-pr and Q-bu exhibited 8.2- and 4.7-fold higher water solubility compared with quercetin, respectively. The in vitro and in vivo studies demonstrated that Q-pr and Q-bu were more effective whereas Q-va was less effective in inhibiting platelet aggregation compared with quercetin. These results indicated that the water solubility and the lipophilicity of the analogues must be improved in order to achieve higher antiplatelet activity, and an optimal acyl chain length is crucial for the synthesized quercetin analogues to be more effective.