The new drug combination of zaltoprofen and paracetamol are widely used as analgesic and antiinfilamatery medication. There is no method reported for simultaneous estimation of this combination for plasma samples. On considering these facts a new reliable, sensitive and accurate reverse phase liquid chromatographic method has been developed for simultaneous quantitative estimation of these drugs in human plasma sample using probinicid as internal standard. In the preliminary screening steps, a factorial design was employed to identify the factors that had significant effects on the selected chromatographic responses. Based on this responses analyses time (tR3), Capacity factor (k1) and selectivity (α) was optimized using Rotatable Central Composite Design (RCCD) and Response Surface Methodology (RSM). Further, this optimized condition was used to analyze the spiked plasma samples, reconstituted by a plasma protein precipitation method. The sample was efficiently separated using Reverse Phase Liquid Chromatography method equipped with PDA detector. The chromatographic separation was carried out using supelcosil LC-8 (150mm × 4.6mm I.D and 5μm particle size) analytical column measured at 254 nm. The developed RP-HPLC method can be applied for the estimation of quantitative pharmacokinetics in preclinical and clinical studies.