This method describes an in vitro assay for measuring INO1 enzyme activity (the conversion of glucose 6-phosphate to myo-inositol 3-phosphate) in cell-free extracts. The method was first described for Plasmodium falciparum cells in MacRae et al. (2014) and consists of two parts: Part 1 describes the assay itself while part 2 describes analysis of the myo-inositol 3-phosphate product using gas chromatography-mass spectrometry (GC-MS).