BackgroundIsoprenoids are a very large class of metabolites playing a key role in plant physiological processes such as growth, stress resistance, fruit flavor, and color. In chloroplasts and chromoplasts, the diterpene compound geranylgeranyl diphosphate (GGPP) is the metabolic precursor required for the biosynthesis of tocopherols, plastoquinones, phylloquinone, chlorophylls, and carotenoids. Despite its key role for the plant metabolism, reports on GGPP physiological concentrations in planta have been extremely scarce.ResultsIn this study, we developed a method to quantify GGPP and its hydrolysis product geranylgeranyl monophosphate (GGP) from tomato fruit, using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). Quantification was done by external calibration and the method was validated in terms of specificity, precision, accuracy, and detection and quantitation limits. We further demonstrate the validity of our approach by analysing GGPP contents in the ripe fruits of wild-type tomatoes and mutants defective in GGPP production. Finally, we also show that the sample preparation is key to prevent GGPP hydrolysis and mitigate its conversion to GGP.ConclusionOur study provides an efficient tool to investigate the metabolic fluxes required for GGPP supply and consumption in tomato fruit.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13007-023-01034-w.