SUMMARYProtein isoforms generated by alternative splicing contribute to proteome diversity. Due to the lack of effective techniques, isoform-specific functions, expression, localization, and signaling mechanisms of endogenous proteinsin vivoare unknown for most genes. Here we report a genetic method, termedisoTarget, for blocking the expression of a targeted isoform without affecting the other isoforms and for conditional tagging the targeted isoform for multi-level analyses in select cells. ApplyingisoTargetto two mutually exclusive isoforms ofDrosophilaDscam, Dscam[TM1] and [TM2], we found that endogenous Dscam[TM1] is localized in dendrites while Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization between Dscam[TM1] and [TM2], rather than any difference in biochemical properties, leads to the two isoforms’ differential contributions to dendrite and axon development. Moreover, withisoTarget, we discovered that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade specifically in axons.isoTargetis an effective technique for studying how alternative splicing enhances proteome complexity.