Acid production and transport are currently being studied to identify new targets for efficient cancer treatment, as subpopulations of tumor cells frequently escape conventional therapy owing to their particularly acidic tumor microenvironment. Heterogeneity in intracellular and extracellular tumor pH (pHi, pHe) has been reported, but none of the methods currently available for measuring tissue pH provides quantitative parameters characterizing pH distribution profiles in tissues. To this intent, we present here a multiparametric, noninvasive approach based on in vivo (31)P nuclear magnetic resonance (NMR) spectroscopy and its application to mouse tumor xenografts. First, localized (31)P NMR spectrum signals of pHi and pHe reporter molecules [inorganic phosphate (Pi) and 3-aminopropylphosphonate (3-APP), respectively] were transformed into pH curves using established algorithms. Although Pi is an endogenous compound, 3-APP had to be injected intraperitoneally. Then, we developed algorithms for the calculation of six to eight quantitative pH parameters from the digital points of each pH curve obtained. For this purpose, each pH distribution profile was approximated as a histogram, and intensities were corrected for the nonlinearity between chemical-shift and pH.