A new, simple and rapid reverse phase-high performance liquid chromatography-diode array detection (RP-HPLC-DAD) method has been developed using sibutramin (S) as internal standard for quantitative determination of guaifenesin (G) and validated both in syrup dosage forms and spiked human plasma samples. The analysis was carried out within 10 min on Inertsil C18 column using isocratic mobile phase consists of phosphate buffer and methanol with pH 3 (60:40, v/v) at a flow-rate of 1 mL min -1 and detection at 212 nm. The concentration-response relationship was linear over a concentration range of 10-100 µg mL -1 for guaifenesin. The developed liquid chromatographic method was successfully applied for the routine analysis of guaifenesin in a cough syrup and human plasma samples without liquid-liquid extraction for quality control purposes. After the method development, bioanalytical method validation was applied. For this purpose, all the validation parameters such as intra-day and inter-day precisions, accuracy, specificity, selectivity and recovery were determined. After the validation procedure, the results were evaluated statistically.