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Cancer is a disease characterized by altered metabolism, and there has been renewed interest in understanding the metabolism of tumors. Even though nutrient availability is a critical determinant of tumor metabolism, there has been little systematic study of the nutrients directly available to cancer cells in the tumor microenvironment. Previous work characterizing the metabolites present in the tumor interstitial fluid has been restricted to the measurement of a small number of nutrients such as glucose and lactate in a limited number of samples. Here we adapt a centrifugation-based method of tumor interstitial fluid isolation readily applicable to a number of sample types and a mass spectrometry-based method for the absolute quantitation of many metabolites in interstitial fluid samples. In this method, tumor interstitial fluid (TIF) is analyzed by liquid chromatography-mass spectrometry (LC/MS) using both isotope dilution and external standard calibration to derive absolute concentrations of targeted metabolites present in interstitial fluid. The use of isotope dilution allows for accurate absolute quantitation of metabolites, as other methods of quantitation are inadequate for determining nutrient concentrations in biological fluids due to matrix effects that alter the apparent concentration of metabolites depending on the composition of the fluid in which they are contained. This method therefore can be applied to measure the absolute concentrations of many metabolites in interstitial fluid from diverse tumor types, as well as most other biological fluids, allowing for characterization of nutrient levels in the microenvironment of solid tumors.