Inositol plays key roles in many cellular processes. Several studies focussed on the quantitative analysis of phosphorylated forms of inositol, enabled by analytical tools developed to detect these highly charged molecules. Direct measurement of free inositol however has been challenging, because the molecule is uncharged and polar. As a result, the mechanisms maintaining the homeostasis of the inositol remains poorly understood. In this study, we overcome these challenges by developing a quantitative liquid chromatography - mass spectrometry (LC-MS) protocol that can resolve and quantify the three main sugar molecules present inside cells: glucose, fructose, and inositol, as well as distinguish the clinically relevant isomers of inositol: myo-, scyllo-, and chiro-inositol. The quantitative power of the new method was validated by accurately monitoring the changes of inositol levels under well-established conditions in Saccharomyces cerevisiae, where the endogenous synthesis of inositol is increased in the transcription repressor OPI1 knockout opi1D and decreased when wild type yeast is fed with exogenous inositol. The method also revealed a new layer of regulation that takes place when exogenous inositol is added to further boost endogenous inositol synthesis in opi1D in a positive feedback loop. Analyses of mammalian cell lines provided many new insights into inositol metabolism. First, different cell lines displayed distinct sugar profiles and inositol concentrations and responded differently to inositol starvation. Second, mammalian cells can synthesize and import scyllo- but not chiro-inositol. Importantly, our method lent direct evidence to the previous hypothesis that lithium treatment could significantly reduce inositol levels in primary cortical neurons, thus diminishing the pool of free inositol available to the phosphoinositide cycle.