Enzymatic parameters are classically determinedin vitro, under reaction conditions that are far from those encountered in cells, casting doubt on their physiological relevance. In this study, we developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parametersin vivo. In the context of a synthetic pathway aiming to produce phytoene and lycopene inSaccharomyces cerevisiae, we focused on four membrane-associated enzymes, a phytoene synthase and three phytoene desaturases, which are difficult to studyin vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration (> 200-fold change) by strategically manipulating the copy number and expression strength of the gene encoding geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determinedin vivo-equivalent Michaelis-Menten parameters (KM, Vmaxandkcat) for GGPP-converting phytoene synthase from quantitative metabolomics, fluxomics and proteomics data, highlighting differences betweenin vivoandin vitroparameters. Then, leveraging the versatility of the same set of strains to cover a wide range of phytoene concentrations, we successfully extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters along a pathway of interest directlyin vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.