We report a method to incorporate a stable isotope (13C) and a radioactive isotope (14C) into a recombinant polypeptide during Escherichia coli culture in M9 minimal medium supplemented with universally labeled 13C- or 14C-labeled glucose. We chose a thermally responsive elastin-like polypeptide (ELP) as a model polypeptide for this study because of its utility in various biotechnology applications such as drug delivery and tissue engineering. High cell densities were obtained by step-wise adaptation of E. coli to M9 medium in addition to supplementing the medium with trace elements that facilitated growth of E. coli. Furthermore, an optimal concentration of isopropyl-β-D-thiogalactopyranoside was determined for induction of ELP expression to achieve high yield (mg/L culture) of the ELP. The incorporation of carbon isotopes was stoichiometrically related to the ratio of labeled glucose to unlabeled glucose in the culture medium. The isotope-labeled variants retained the physicochemical properties of the unlabeled ELP, specifically its temperature dependent aggregation behavior. As an example of the utility of this method, the in vitro stability of 14C-labeled ELP in PBS and mouse serum was conveniently quantified by SDS–PAGE and autoradiography. In addition, the in vivo stability of the 14C-labeled ELP in plasma was determined along with its plasma pharmacokinetics.