Cancer cell growth is predicted to require substantial increases in the rates of substrate catabolism and ATP turnover to drive biosynthesis and cell growth. Strategic limitation of substrate supply is therefore a potentially effective way to slow tumor growth. However, the effects of substrate limitation on the rates of ATP production and consumption, especially over the acute timescales relevant to most experimental design, are not straightforward. First, bioenergetic homeostasis will oppose substrate limitation by increasing catabolism of substrates that remain accessible, in order to meet existing ATP demand. Second, ATP demand itself may be different under different substrate conditions. Empirical evaluation of total cellular ATP production is therefore required. Cultured cells remain an important model system for understanding these mechanisms of bioenergetic control.Using extracellular flux analysis to estimate total cellular ATP production rate, we demonstrate that the MCF7 breast cancer model supports rates of basal ATP production over an approximately 2‐fold range, in the presence of the single substrates glucose, glutamine, and pyruvate, the main substrates commonly present in standard culture media. In comparison, a nontransformed cell culture model shows maintenance of a single ATP production rate across the same single substrates. These novel findings demonstrate that MCF7 cells are highly flexible with respect to total cellular ATP production, over a timeframe that is independent of the slower cellular remodeling caused by altered transcriptional regulation.Support or Funding InformationNIH 1R15ES025917‐01A1; Touro University California College of Pharmacy