Carotenoids are important secondary metabolites, which have been recognized as an essential component of the human diet because of their valuable beneficial health effects. With this rationale, there is a continuous aim to define the distribution of these compounds in plants, to better understand their metabolism and to increase their concentration levels in fruits and vegetables. This study aimed at deepening the knowledge on the regulatory role of phytohormones in carotenoid metabolism. More specifically, it was envisaged to reveal the phytohormones involved in the metabolism of a-carotene, b-carotene, lycopene, lutein and zeaxanthin. To this purpose, the phytohormone profiles of 50 tomato fruits were determined by high-resolution Orbitrap mass spectrometry and evaluated towards the associated carotenoid levels. Data mining was performed by differential expression and orthogonal partial least squares analyses. This metabolomics approach revealed 5 phytohormonal metabolites, which significantly influenced (Variable Importance in Projection scores C0.80) carotenoid metabolism. These metabolites were identified as cis-12-oxo-phytodienoic acid, cucurbic acid, 2-oxindole-3-acetic acid, 1-acetylindole-3-carboxaldehyde, and cis-zeatin-O-glucoside. The involvement of the individual phytohormones towards carotenoid metabolism was investigated by regression analysis (P values B0.05, R2 varying between 0.280 and 0.760) and statistical correlation (P values B0.01, correlation varying between 0.403 and 0.846). It was concluded that these phytohormones all have significant contributing value in the regulation of carotenoid metabolism, thereby exhibiting down- and upregulating influences. As a result, this knowledge encloses the potential for improving tomato fruit nutritional quality by targeted control of agronomic conditions, exogenous use of plant bioregulators, or genetic engineering.