This protocol enables transcriptome profiling of specific cell or tissue types that are isolated from tomato using laser microdissection (LM). To prepare tissue for LM, fruit samples are first fixed in optimal cutting temperature (OCT) medium and frozen in molds. The tissue is then sectioned using a cryostat before being dissected using an LM instrument. The RNAs contained in the harvested cells are purified and subjected to two rounds of amplification to yield sufficient quantities of RNA to generate cDNA libraries. Unlike several other techniques that are used to isolate specific cell types, LM has the advantage of being readily applied to any plant species without having to generate transgenic plants. Using the protocols described here, LM-mediated cell-type transcriptomic analysis of two samples requires ~8 d from tissue harvest to RNA sequencing (RNA-seq), whereas each additional sample, up to a total of 12 samples, requires ~1 additional day for the LM step. RNA obtained using this method has been successfully used for deep-coverage transcriptome profiling, which is a particularly effective strategy for identifying genes that are differentially expressed between cell or tissue types.