AbstractUbiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes. The Skp1–Cullin–F‐box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F‐box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F‐box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F‐box proteins in the SCF complexes affinity‐purified en masse from budding yeast cells at log and post‐diauxic phases, and revealed an increment of Saf1, an F‐box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F‐box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex‐mediated ubiquitination system. Compositional alteration of incorporated F‐box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis.