Precise cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach whereby separate translational reading frames are coupled to the inclusion or exclusion of cell-specific alternative exons. Candidate exons are identified by analyzing publicly available RNA sequencing datasets and filtering by cell specificity, sequence conservation, and intron length. This method, which we term splicing-linked expression design (SLED), can be combined in a Boolean manner with existing minipromoters and viral serotypes. We generated AAV-based SLED vectors that selectively target all neurons, photoreceptors, or excitatory neurons. We further used SLED to generate a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show the translational potential of SLED by rescuing photoreceptor degeneration in Prph2rds/rds mice and by developing an oncolytic vector that can selectively induce apoptosis in SF3B1 mutant cancer cells. The flexibility of SLED technology enables new avenues for basic and translational research.