Studies of the topology, functioning, and regulation of metabolic systems are based on two main types of information that can be measured by mass spectrometry the (absolute or relative) concentration of metabolites and their isotope incorporation in C-labeling experiments. These data are currently obtained from two independent experiments because the C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the C-mass fractions from which its C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two sets of information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of 2 both the number of cultivations to perform and the number of samples to collect, prepare, and analyze. The proposed approach is based on an IS labeled with other isotope(s) that can be resolved from the C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled NC-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.