Introduction: Technetium-99m pertechnetate is the most readily available radionuclide employed for thyroid uptake using gamma camera, which provides an assessment of the trapping ability of the gland. The present study was carried out to compare Tc-99m pertechnetate thyroid uptake values by three different protocols (gamma camera, uptake probe using computer software and uptake probe using manual method). Material and Methods: This prospective study was performed in twenty seven consecutive patients (5 males and 22 females) using three different protocols and two different modalities ie. Gamma camera and uptake probe. After intravenous administration the mean time taken for the uptake on gamma camera and thyroid uptake probe was 30 ± 10.02 minutes (range 20- 52 minutes) and 32.1± 12.65 minutes (range 21-75 minutes). Results: The mean uptake values and standard deviation calculated using thyroid uptake probe with computer software and Manual method (8.1±6.1, and 8.06±5.9) were slightly higher than uptake values calculated using Gamma camera (5.8±6.2). The correlation (r) between probe and manual uptake, gamma camera and manual uptake, and gamma camera and probe were 0.9947, 0.9691 and 0.9625 respectively. The correlation coefficient between all the three uptake methods is very high and shows a positive trend. Authors observed that the uptake values evaluated by the two modalities co-relate well but their mean values are different significantly. However, there is not much difference between the uptake values calculated using the probe software and the manual calculation formula. Conclusion: The uptake probe can be routinely used for thyroid uptake calculation using Tc-99m pertechnetate. But standardization of the thyroid uptake using pertechnetate needs to be performed before putting this for patient’s routine use. The gamma camera underestimates the thyroid uptake than the probe but provides the thyroid image for the size and shape evaluation.