Honokiol, a biphenolic neolignan isolated from Magnolia officinalis , was reported to have a promising anti-inflammatory activity for the treatment of various diseases. There are many efforts on the synthesis and structure–activity relationship of honokiol derivatives. However, regioselective O -alkylation of honokiol remains a challenge and serves as a tool to provide not only some derivatives but also chemical probes for target identification and mode of action. In this study, we examined the reaction condition for regioselective O -alkylation, in which C2 and C4′-alkylated analogs of honokiol were synthesized and evaluated for inhibitory activity on nitric oxide production and cyclooxygenase-2 expression. Furthermore, we successfully synthesized a potential photoaffinity probe consisting of biotin and benzophenone based on a C4′-alkylated derivative.