Overnight Express™ Autoinduction NMR-¹⁵N, -¹³C, when supplemented with ¹³C-labeled glycerol, provides reagents to make 1 L of defined autoinducing liquid culture medium used to produce [U-¹⁵N, U-¹³C]-labeled proteins for NMR spectroscopy. The autoinduction method is based on the differential metabolism of carbon sources to promote growth to high cell density and to automatically induce protein expression under control of lac promoters, eliminating the need to monitor cell densities or add inducer (IPTG). The system includes four concentrated sterile solutions: OnEx Dual-NMR Solution 1 is a blend of carbon sources optimized for tightly regulated uninduced growth to high density, followed by induction; OnEx NMR Solution 2 is a buffer and nitrogen blend that mediates metabolic acid production and provides additional nitrogen for increased protein synthesis; OnEx Solution 3 provides magnesium for maximal cell density; OnEx Solution 4 provides trace metals to minimize growth limitations associated with mineral deficiencies and satisfy the requirements of metal-containing target proteins, even at high expression levels. The kit also includes ¹⁵N-labeled solid ammonium chloride, ¹³C-labeled solid glucose, and reagents to make a non-inducing starter medium, for staged growth without expression and for stable storage of clones. Efficient ¹³C protein labeling requires ¹³C-glycerol, which must be purchased separately. Addition of the four liquid components and sterile solutions of ¹⁵N ammonium chloride, ¹³C glucose, and ¹³C glycerol to sterile water results in a chemically- defined medium promoting high cell densities, autoinduction of expression, and efficient labeling of target proteins.