In many medical test designs, presence of a color spot can represent existence of a disease. This presence is usually verified by observation. For these tests, knowing the color discrimination threshold is necessary for modifying the indicator spots to have color differences above the threshold. In this work, a psychophysical experiment is used to determine the color difference threshold for a veterinarian test device from IDEXX company with blue-green spots. The study was conducted in two phases, In the first phase a preliminary investigation was conducted for the ideal situation, that is having perfect circular spots without any noise or non-uniformity. Method of constant stimuli was used to present designed test images to the observers. The results were analyzed using Probit analysis method. The second phase of the study was performed with objective of studying the effects of noise, imperfect spot shapes, presence of streak and presence of spot color gradient on the color difference threshold between the background and spot colors. The same experimental and analysis method was used in both phases. The results for the ideal situation showed an average discrimination threshold of 1.27 color difference (DE00) for overall data. For the realistic situation, the noisy appearance of the image and imperfectness of the shape of the spots did not affect the threshold when observers were expecting imperfect spots. However, the presence of streak and spot gradient increased the threshold.