The present study describes the application of concept of sample pooling to increase the throughput of pharmacokinetic screening at drug discovery and development stage. An HPLC-UV method for the simultaneous estimation of three synthetic antimalarial compounds 99/357, 99/408 and 99/411 has been developed and validated in rat serum with internal standard for pharmacokinetic profiling. Drug compounds in serum were extracted by two-step liquid-liquid extraction with 2% isopropyl alcohol in n-hexane and quantitated using a validated gradient HPLC-UV method, which was made feasible for all compounds using gradient elution scheme. The method was validated in terms of HPLC reproducibility, linearity, specificity, recovery, accuracy and precision, freeze thaw stability and long-term storage stability. Excellent linear relationships (r>0.99) were obtained for calibration as well as analytical standards over a concentration range of 25-1000 ng/ml for three analytes. Recoveries were fond to be >85% for 99/408 and 99/357 and >70% for 99/411. The method developed for three analytes was found to be accurate and precise as bias and percent relative standard deviation (% R.S.D.) values were within limits (<20%). By employing sample pooling approach, plasma level - time profile following single intravenous dose of all three compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.