Bioanalytical methods of Reverse Phase-High performance liquid chromatography (HPLC) was developed and validated for simultaneous estimation of Isoniazid (INH) and Ciprofloxacin Hydrochloride (CIP HCl) encapsulated in lipid polymeric hybrid nanoparticles (LPNs) in plasma and in organ homogenates of lung, liver, spleen and kidney of mice. Chromatographic separation was done using Agilent® C18 bonded silica column of dimensions 150 × 4.6 mm, 5μmwith flow rate 1.0 ml/min, injection volume 20 µland column temperature 40°C. The mobile phase consists of a mixture of 70 volumes of 0.1 percent v/v of trifluoroacetic acid(TFA) and 30 volumes of acetonitrile (ACN). The results indicated that the developed method was linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with relative standard deviation values lower than 5, which are in accordance with USFDA guidelines for bioanalytical method validation. Isoniazid (INH) and Ciprofloxacin Hydrochloride (CIP HCl) were stable in plasma and tissue homogenates under different storage and processing conditions. This method was applied to study the pharmacokinetic and biodistribution profile of both drugs in free form and in bound state with lipid nanoparticles. The results showed that polymeric nanoparticles showed higher drug accumulation in the target site i.e. lung as compared to non-target organs fulfilling our aim of developing a HPLC method for the simultaneous estimation of both drugs and their application in determination of pharmacokinetic and pharmacodynamic potential of the lipid nanoparticles.