Selenoproteins, in which the selenium atom is present in the rare amino acid selenocysteine, are vital components of cell homeostasis, antioxidant defense, and cell signaling in mammals. The expression of the selenoproteome, composed of 25 selenoprotein genes, is strongly controlled by the selenium status of the body, which is a corollary of selenium availability in the food diet. Here, we present an alternative strategy for the use of the radioactive 75Se isotope in order to characterize the selenoproteome regulation based on (i) the selective labeling of the cellular selenocompounds with non-radioactive selenium isotopes (76Se, 77Se) and (ii) the detection of the isotopic enrichment of the selenoproteins using size-exclusion chromatography followed by inductively coupled plasma mass spectrometry detection. The reliability of our strategy is further confirmed by western blots with distinct selenoprotein-specific antibodies. Using our strategy, we characterized the hierarchy of the selenoproteome regulation in dose–response and kinetic experiments.