Recent advances of image-based in situ mRNA quantification methods allow to visualize where in a tissue section a set of genes is expressed. It enables to map large numbers of genes in parallel and by capturing cellular boundaries allows to assign genes to cells. Here, we present a high-throughput, multi-targeted gene expression profiling technique called in situ sequencing that is capable of localizing hundreds of genes simultaneously and supports cell type classifications that follow transcriptome-based taxonomy. In situ sequencing is a targeted, amplified, and barcoded approach using padlock probes (PLPs) and rolling circle amplification (RCA). The current protocol relies on mRNA fixation, mRNA reverse transcription, residual mRNA degradation, and PLP hybridization. PLPs are amplified by RCA and labeled with fluorophore-conjugated probes, allowing their detection under conventional fluorescence microscopes.