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Crystals isolated from Hylocereus polyrhizus were analyzed using four different approaches—X-ray Crystallography, High Performance Liquid Chromatography (HPLC), Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Nuclear Magnetic Resonance (NMR) and identified as myo-inositol. The X-ray crystallography analysis showed that the unit-cell parameters were: a = 6.6226 (3) Å, b = 12.0462 (5) Å, c = 18.8942 (8) Å, α = 90.00, β = 93.98, δ = 90.00. The purity of the crystals were checked using HPLC, whereupon a clean single peak was obtained at 4.8 min with a peak area of 41232 μV*s. The LC-MS/MS technique, which is highly sensitive and selective, was used to provide a comparison of the isolated crystals with a myo-inositol standard where the results gave an identical match for both precursor and product ions. NMR was employed to confirm the molecular structure and conformation of the crystals, and the results were in agreement with the earlier results in this study. The discovery of myo-inositol crystals in substantial amount in H. polyrhizus has thus far not been reported and this is an important finding which will increase the marketability and importance of H. polyrhizus as a crop with a wide array of health properties.