Protein labeling with stable isotopes such as ²H, ¹³C, ¹⁵N, and ¹⁸O is an important tool for structural and bioanalytical methods such as nuclear magnetic resonance (NMR), small angle neutron scattering (SANS), and mass spectrometry. Although protocols have existed to express isotope labeled proteins in E. coli for years, a large number of open questions remain unanswered, such as why cells must be acclimated for growth in ²H, what the effect of heavy isotopes is on cell physiology, etc. Moreover, stable isotope labeling of proteins using yeast and mammalian cell systems is only at its infancy, with very few studies reported. Our ongoing research is aimed at understanding the mechanism of E. coli, yeast, and mammalian cell adaptation to growth in the presence of heavy isotopes, and to use this knowledge to develop tools to enable more routine high level production of labeled proteins using these different expression platforms.