Multidrug therapy has become the standard treatment of acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV). Nucleoside reverse transcriptase inhibitors (NRTIs) are used in all AIDS combination therapies. In combination therapies, there is a high potential for pharmacokinetic interaction between the individual drugs. This interaction can alter the transport profile of anti HIV agents to the fetus in pregnant women. In order to study pharmacokinetic interactions, sensitive and valid analytical methods are required for the quantification of NRTIs in different biological matrices. This dissertation focuses on developing valid analytical methods to quantify NRTIs in different pregnant rat matrices. Lamivudine (3TC) and zidovudine (AZT) were studied as model NRTIs. These matrices include plasma, amniotic fluid, placenta and fetus. Method development includes 3 steps, sample preparation, chromatographic separation and spectroscopic detection. Due to the complicity of tissue matrices, ultra clean sample extraction is required. Different sample preparation techniques like protein precipitation by acids, organic solvents and salting out, solid phase extraction (SPE) and liquid-liquid extraction (LLE) were used. High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are the separation techniques that were used. For spectrometric detection, ultraviolet (UV) and mass spectrometry (MS) detectors were used. After developing the optimum sample extraction, chromatographic separation and spectrometric detection conditions, the methods were validated according to FDA criteria. The validated methods were successfully applied in animal studies using the pregnant rat model. The animal studies have shown that fetal exposure to 3TC was significantly increased when co-administered with AZT. The mechanism of this drug-drug interaction has not been found, but it may be due to AZT competitive inhibition of the 3TC efflux transporters in the fetal-facing side of the placenta. These results suggest that the underlying mechanism behind the 3TC placental transport in rats is carrier mediated.