We developed a method for monitoring energy expenditure of tissue biopsies using the cell culture viability reagent AlamarBlue to assess reduction potential (NADH, NADPH, F ADH₂, and FMNH₂) within the organism or tissue sample. We applied this assay to compare the metabolic rate of biopsies taken from a variety of mouse tissues and predict milk production. Tissue biopsies (<10 mg) of murine heart, kidney, liver, brown adipose, red skeletal muscle and white skeletal muscle were collected into wells of a 96-well plate containing DMEM without Phenol Red (4.5 g/L glucose) immediately following decapitation under bell jar isoflurane anesthesia. Fluorescence (ex: 530 nm, em: 590 nm) was measured on a SpectraMax M2 plate reader at tissue collection and 1, 2, and 4 h later. Plates were incubated in 5% CO₂ at 37^?C between measurements. The change in fluorescence/mg tissue increased linearly with time to 4h, suggesting that the tissue remained viable throughout the experiment. Fluorescence change/mg tissue which varied up to 8.9 times was highest in kidney, and in descending order the remaining tissues included brown adipose tissue, liver, heart, red skeletal muscle, and white skeletal muscle (P < 0.0001). We subsequently, tested the application of this assay to assess mammary gland metabolic function, while concurrently measuring ex vivo lactose production. On the day of parturition, mammary glands were collected after sacrifice, and assessed for ex vivo lactose production and metabolic rate. To induce variability in ex vivo lactose production, mice were exposed to differing environmental conditions during the final 5 d of gestation (thermoneutral environment (22°C, 50% relative humidity), heat stress (HS; 35°C, 50% relative humidity) and thermoneutral pair-feeding (PF). Heat stress decreased lactose production (46%; P < 0.05) and fluorescence change (36%; P < 0.05) relative to PF control mice. Moreover, ex vivo metabolic rate and lactose production correlated linearly (R² = 0.68, P = 0.0035). Combined these studies show that this assay can be applied to assess metabolic rate across several tissues, assess the effect of physiological perturbations on metabolic rate in a given tissue, and predict production.