There are different types of radiation detectors (gas-filled, semiconductor, scintillator etc.) and various working principles (ionization, electron-hole pair production, excitation) used in radiation detection. It is important that any detector is selected according to its intended use and that the detector is operated under appropriate conditions. NaI(Tl) scintillation detectors are widely used in nuclear spectroscopic studies due to their known advantages. Prior to carrying out such studies, it is first necessary to know the response function of the detector against radiation under the expected operating conditions and to perform the appropriate calibrations. It is important that certain factors are optimized, such as the experimental setup (source-to-detector position, shielding, source activity etc.), gain, bias voltage, and shaping time, which can each significantly affect the response function. In addition, in computational studies based on experimentation, optimization is gain more significant since it is assumed that such experimental studies are carried out under optimum conditions. In this study, in order to determine the most suitable operating conditions for a 3 × 3 inch NaI(Tl) detector; a coaxial sample holder was manufactured to enable different source-to-detector distances (5–15 cm) to be tested, and spectra were then obtained using different bias voltages (750–850 V) and sources with different energies in the range of 81–1332 keV. Through the use of efficiency values obtained for each condition, line and 3D surface plots were drawn and the relations between efficiency and distance, energy and bias voltage examined. The results showed that the efficiency value had an exponentially decreasing characteristic that was dependent on the energy and source-detector distance, and had the highest value at a bias voltage of around 800 V.