Cells utilize myo-inositol for osmoregulation and phosphatidylinositol signaling. Mass spectrometric and in vivo magnetic resonance spectroscopic techniques have been complementarily used in our laboratories to investigate brain myo-inositol metabolism. Mass spectrometric quantitation methods are surveyed focusing primarily on derivatization reactions, gas chromatographic separation and detection of ions. Monitoring of the m/z 373 fragment ion generated from acetate derivative provides precise quantitation of myo-inositol in biological matrices. The technique and its clinical applications are discussed. Measurement of myo-inositol transport using a stable isotope technique is illustrated for cultured neurons. In addition, the possible use of the technique in probing phosphatidylinositol turnover is discussed. An in vivo 1H magnetic resonance spectroscopic technique is described for measuring the absolute concentration of myo-inositol in human brain. Magnetic resonance spectroscopy with short echo-time enables detection of the resonance peak of myo-inositol (3.56 ppm) when the water resonance peak is suppressed by narrow band radio-frequency pulses. The review focuses on an external reference method involving collection of data from the human subject and the phantom containing aqueous myo-inositol standard solution in the same scanning session. The method takes into account differences in longitudinal and transverse relaxation time constants of myo-inositol between brain tissue and aqueous solution. Application of the technique is illustrated by measuring brain myo-inositol in Down syndrome adults and Alzheimer disease patients. Advantages and limitations of this noninvasive technique in monitoring metabolic processes are discussed.