The comparative pharmacokinetic study or levodopa (l-dopa) nasal microspheres (test) and l-dopa carbidopa (c-dopa) nasal formulation (reference) was carried out on 120 healthy experimental rats in 20 groups. The concentration of l-dopa in brain was determined by LC/MS-MS. All pharmacokinetic parameters were calculated using a non- compartmental model. 90% bootstrap confidence interval (CI) of test/reference AUC and Cmax ratio were calculated to access bioavailability. The ratio and 90% CI for nasal l-dopa/nasal l-dopa+c-dopa formulation was 96.94 (60.05-133.83) for Cmax and 92.29 (66.54-118.04) for AUCt. Despite 90% CI limit not within the bioequivalence limit of 80% to 125% both formulation are equivalent at ratio test. The above statement is further proved by student t-test as probability of detecting difference between above formulations, at 95% CI was 0.547 which signifies there is no significant difference between two formulations. Therefore it can be concluded that bioadhesive nasal microspheres containing l-dopa alone can be used in treatment of Parkinson's disease. © 2015 Scientific Publishers. All Rights Reserved.