Background:The goal of proposed study was to optimize and validate reversed phase high performance liquid chromatography (RP-HPLC) method for assessment of eluxadoline in presence of its forced degradation products in in-house tablet mixture. Materials and Methods: RP-HPLC method was developed using SunQSil C 18 (250 mm × 4.6 mm, 5 µm) as stationary phase and 20 mM sodium acetate: methanol (45:55, v/v) as mobile phase at a 1 ml/min flow rate with 205 nm detection wavelength. The method was found to give a resolved peak of eluxadoline at retention time of 5.32 min. To study degradation behaviour of eluxadoline, it was exposed to acid hydrolysis, alkali hydrolysis, oxidation, photolysis and thermolysis. The developed method was suitable to resolve eluxadoline from its all-degradation products. Validation of method was carried out in accoradance with ICH quality guideline Q2(R1). Results: In the concentration range of 3 -18 µg/ml, the linearity of the proposed process was observed with regression coefficient (R 2 ) of 0.9987. Sensitivity parameters, LOD and LOQ were observed to be 0.8627 µg/ml and 2.6144 µg/ml, respectively. In accuracy studies, the recovery of the drug was found to be between 99.1956-100.7294%. It was noticed that the developed was precise as the values of % RSD were found to be less than 2.0 % for both intraday and interday precision. The method was robust and can be useful for regular analysis of formulations containing eluxadoline. Conclusion: The developed method was utilised for determination of content of eluxadoline in in-house tablet mixture.