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Purpose: The aim of the present study was to develop a simple and accurate way to measure vitamin D levels. Vitamin D nowadays is measured by a variety of methods which their common drawbacks are expensive equipment and the need for high trained technical staff. In this research we measured vitamin D levels by means of Fourier transform infra red method in conjugation with the evanescent wave spectroscopy technique, in order to develop a simpler vitamin D measurement method. Methods: Blood samples were collected from patients with vitamin D deficiency at five intervals before and up to 16 days after they took a dose of 200,000 IU vitamin D 3. Samples were measured by the conventional bio-chemical method and by the evanescent wave spectroscopy means. Results: Correlation was found between the vitamin D levels measured by the traditional method and by the evanescent wave spectroscopy technique. The absorption lines occurred prominently in the IR spectral regions of the Amide I (&1650 cm -1),Amide II (&1530 cm -1) and the (&3400 cm -1) absorption band which is attributed to the hydroxyl group indicated by the O-H stretch. In addition, the examination of the blood samples using the evanescent wave spectroscopy with clustering techniques facilitated the discrimination between vitamin D deficiency and normal vitamin D levels. Practical relevance: This study demonstrates the potential of using the Fourier transform infra red method in conjugation with the evanescent wave spectroscopy techniquecoupled with multivariate analysis as a non-expensive, rapid and accurate alternative to the routine methodologies.