Eluxadoline is a novel μ- and κ-opioid receptor (OR) agonist and δ-OR antagonist, recently approved as a first line therapy for the treatment of irritable bowel syndrome. Due to abuse potential, poor bioavailability and high intersubject variability, a sensitive and reliable assay is prerequisite for its determination in biological samples. This work first time report the development and validation of UPLC–MS/MS assay for determination of eluxadoline in rat plasma sample using risperidone as an internal standard (IS). A high-throughput 96-well plate format μ-SPE technique was used for plasma sample extraction. The extracted samples were separated on Acquity BEH™ C18 column (100 × 2.1 mm, 1.7 μm) using mobile phase elution of acetonitrile: 20 mM ammonium acetate (80:20, v/v) at a flow rate of 0.3 mL min−1. The precursor to product ion transition of m/z 570.16 → 118.12 (qualifier), 570.16 → 171.08 (quantifier) for eluxadoline, and m/z 411.18 → 191.07 for IS were used for MRM monitoring. The calibration curves were linear in concentration range of 0.15–50 ngmL−1 with LOD and LOQ of 0.07 and 0.15 ngmL−1, respectively. The validation results satisfied the criteria of USFDA and SWGTOX guidelines and were within the acceptable limit. Finally, the method was successfully applied in bioavailability enhancement study of the newly developed PLGA nanoparticles and Eudragit coated PLGA nanoparticles of eluxadoline in rats.