The development of label-free optical biosensors is motivated by the need for highly accurate and sensitive measurements of biomolecular interactions. The Interferometric Reflectance Imaging Sensor (IRIS) delivers precise and multiplexed detection of such interactions. A significant challenge in label-free sensing is the bulk effect, which is the presence of unwanted signals caused by variations in refractive index that can obscure true binding interactions and lead to inaccurate measurements. This thesis presents multiple advancements to IRIS technology focusing on the quantitative detection and elimination of the bulk effect using the principles of light reflection in different colors. A novel bulk-effect-free signal calculation method is introduced, significantly reducing sensitivity to refractive index variations. Additionally, a methodology for real-time detection of changes in refractive index is developed. Both systems are theoretically validated through MATLAB simulations. Experiments were conducted to demonstrate the effectiveness of the bulk-effect- free signal measurement and the refractive index detection system. Two main types of experiments were performed: with solutions of varying refractive indices without actual binding to detect refractive index changes and binding experiments to test new systems' effectiveness in detecting true biomolecular interactions. Novel experimental procedures using a combination of these methods were introduced to eliminate the bulk effect. This thesis establishes the foundation for the next-generation multicolor IRIS system, enhancing its potential for accurately detecting biomolecular interactions by eliminating the bulk effect and incorporating refractive index detection. / 2026-09-10T00:00:00Z