Lifitegrast is an LFA-1 antagonist, tetrahydroisoquinoline derivative, formulated as sterile eyedrops. Lifitegrast ophthalmic solution 5.0% was the first medication approved by the US FDA for treatment of the signs and symptoms of DED. We developed and validated two novel, economic, specific, and sensitive UV spectrophotometric and RP-HPLC methods for estimation of lifitegrast in bulk and dosage form. The linearity was found in the concentration range of 05-30 μg/mL in UV method (R²=0.9995) and 2-12 μg/mL in HPLC method (R²=0.999) with good correlation coefficient. The LOD and LOQ were found to be 0.77 μg/mL and 2.33 μg/mL, respectively by UV method. RP-HPLC method was developed at SunFire C 18 column (250 × 4.6 mm i.d., 5μm) using methanol, acetonitrile, and water as a mobile phase in the ratio of 20:60:20 (v/v) (pH = 2.27 adjusted with orthophosphoric acid). The LOD and LOQ were found to be 0.50 μg/mL and 1.52 μg/mL respectively by HPLC method. These developed methods were validated according to ICH (Q2 (R1)) guidelines with the following validation parameters i.e., specificity, linearity, accuracy, precision, LOD, LOQ, robustness, and ruggedness. The results of the study proved the applicability of the method in routine analysis of lifitegrast.