Thyroid nodules are common and often require fine needle aspiration biopsy (FNAB) to determine the presence of malignancy to direct therapy. Unfortunately, approximately 15-30% of thyroid nodules evaluated by FNAB are not clearly benign or malignant by cytology alone. These patients require surgery for the purpose of diagnosis alone; most of these nodules ultimately prove to be benign. Elastic light scattering spectroscopy (ESS) that measures the spectral differences between benign and malignant thyroid nodules has shown promise in improving preoperative determination of benign status of thyroid nodules. We describe the results of a large, prospective, blinded study validating the ESS algorithm in patients with thyroid nodules. An ESS system was used to acquire spectra from human thyroid tissue. Spectroscopic results were compared to the histopathology of the biopsy samples. Sensitivity and specificity of the ESS system in the differentiation of benign from malignant thyroid nodules are 74% and 90% respectively, with a negative predictive value of 97%. These data suggest that ESS has the potential for use in real time diagnosis of thyroid nodules as an adjunct to FNAB cytology.