Eluxadoline, a mixed opioid receptor modulator, is used for the treatment of irritable bowel syndrome with diarrhea (IBS-D). However, its systemic metabolism and potential adverse effects necessitate a colon-targeted drug delivery approach to enhance therapeutic efficacy while minimizing off-target effects. This study aims to develop, optimize, and characterize a colon-targeted nanoformulation of eluxadoline using a factorial design approach for improved localized drug delivery, enhanced bioavailability, and therapeutic efficacy in IBS treatment. A factorial design-based optimization strategy was employed to develop eluxadoline-loaded nanoparticles with desirable particle size, entrapment efficiency, and drug release profile. The formulation was characterized using particle size analysis, zeta potential measurement, drug loading efficiency, and surface morphology evaluation.