Thyroid uptake study is a technique that requires injection of a radio-isotope/radiotracer emitting gamma rays into the blood stream of the patient. Thyroid imaging is done by means of Thyroid Uptake imaging system. In the absence of a sophisticated system, imaging can also be done using Gamma Camera. By intravenously injecting 2 millicuries of Technetium-99m pertechnetate radio-isotope, serial thyroid images are acquired. This Uptake study provides Functional information and is useful for diagnosis and treatment of Hyperthyroidism. Thyroid uptake study done using Gamma Camera has to be calibrated at each laboratory using this technique. In our hospital it has been standardized that a tracer uptake of greater than 2 % is considered Hyperthyroidism, between 0.5 and 2 % is considered normal and less than 0.5 % is considered Hypothyroidism. Thyroid Uptake is calculated based on the counts. Counts are nothing but the sum of all intensities in the selected region of the image. Gamma Camera uses a LEAP (Low Energy All Purpose) collimator which handles only photons emitted from radioisotopes having lower emission energies. So Technetium-99m is used which has its energy of emission around 140 keV. For a typical Thyroid Uptake Probe where Iodine-131 having greater emission energy of 364 keV is preferred, existing Thyroid Uptake software cannot be used. Therefore an Interactive GUI (Graphical User Interface) tool was developed for thyroid Uptake studies using Fiji for determination of tracer uptake by manually drawing the ROI (Region of Interest) around left and right thyroid lobes separately. Developed tool was tested on 30 real time thyroid cases (26 female and 4 male) and the uptake values obtained are compared with those obtained from the existing software tool.Index Terms -Interactive GUI, Fiji, ROI, Anticubital, Radiotracer.