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Plant organs and tissues are comprised of an array of cell types often superimposed on a gradient of developmental stages. As a result, the ability to analyze and understand the synthesis, metabolism, and accumulation of plant biomolecules requires improved methods for cell- and tissue-specific analysis. Tomato (Solanum lycopersicum) is the world’s most valuable fruit crop and is an important source of health-promoting dietary compounds, including carotenoids. Furthermore, tomato possesses unique genetic activities at the cell and tissue levels, making it an ideal system for tissue- and cell-type analysis of important biochemicals. A sample preparation workflow was developed for cell-type-specific carotenoid analysis in tomato fruit samples. Protocols for hyperspectral imaging of tomato fruit samples, cryoembedding and sectioning of pericarp tissue, laser microdissection of specific cell types, metabolite extraction using cell wall digestion enzymes and pressure cycling, and carotenoid quantification by supercritical fluid chromatography were optimized and integrated into a working protocol. The workflow was applied to quantify carotenoids in the cuticle and noncuticle component of the tomato pericarp during fruit development from the initial ripening to full ripe stages. Carotenoids were extracted and quantified from cell volumes less than 10 nL. This workflow for cell-type-specific metabolite extraction and quantification can be adapted for the analysis of diverse metabolites, cell types, and organisms