A single bolus dose of theophylline (Th; 5 mg/kg) was administered through the abdominal catheter in nonpregnant and pregnant rats to measure unbound fraction (fu) of Th in pregnancy. After 90 min blood samples were collected. Plasma was extracted with a solvent system comprising chloroform and isopropyl alcohol (IPA) followed by iso-cratic HPLC analysis. Results showed an increase in unbound fraction of Th in late pregnancy.