A Ru(II)-catalyzed regioselective C-H activation toward hydroxymethylation of β-carbolines and isoquinolines as effective directing groups has been developed, and the mechanism was probed by using online electrospray ionization-tandem mass spectrometry. The introduction of the hydroxymethyl group in the biologically relevant molecules routed via C-H functionalization remains an important task. Gratifyingly, this protocol draws attention to the regioselective formation of monohydroxymethylated β-carboline/isoquinoline products exclusively.