The pupil, a small opening in the center of the iris, not only regulates the amount of light entering the eye but also acts as a critical indicator of ocular health and nervous system function. Often, its shape is not perfectly circular or elliptical, which adds complexity to its analysis. In this study, an advanced hardware and software system was developed to automate the calculation and analysis of pupillary responses to RGB isoluminant stimuli. This innovative system allows the analysis of pupillary response variability by employing image processing algorithms to track pupillary reflexes accurately. This approach provides a valuable tool for better understanding behavioral patterns in pupillary dynamics in response to RGB chromatic light stimuli of identical luminance. In addition, before the execution of this project, a research protocol was submitted to the UAEM Research Ethics Committee for evaluation and approval, adhering to the principles outlined in the Declaration of Helsinki. The protocol was registered under number 007.2023.