Doxycycline Hyclate (DOX) is an antibacterial drug which is member of the second tetracycline group and the absorption of DOX is reduced about 20% in the presence of skimmed milk. Therefore new drug delivery system can be advisable for DOX to reduce the food and drug interaction and improve the bioavailability. The aim of the present study is to prepare a microemulsion system of DOX which could result in reducing in food interaction and an improvement of oral bioavailability by increasing the drug's permeability. For this purposes, DOX was radiolabeled with Tc-99m. Radiochemical purity was determined with radioactive thin layer chromatography (RTLC) studies. Permeability of DOX from Tc-99m-DOX solution (Tc-99m-DOX-S) and Tc-99m-DOX loaded microemulsion (Tc-99m-DOX-M) was investigated with in vitro cell culture studies by using human colonic adenocarcinoma cell line (Caco-2). The radioactivity of Tc-99m-DOX-M for the apical to basolateral direction (Papp (A -> B)) and basolateral to apical direction (Papp (B -> A)) were found higher than Tc-99m-DOX-S. Based on the in vitro cell culture studies, this dosage form is a promising formulation as an alternative for oral drug delivery of DOX.