Eugenol is an important phytochemical bioactive compound of ayurvedic and other marketed herbal formulations. It shows anti-inflammatory, anti-bacterial and anti-tubercular activity. Thus, it is a suitable bioactive biomarker to establish the quality of commercial drug and its formulation. The aim is to develop and validate an efficient and effective RP-HPLC method for quantification of eugenol from commercial marketed formulations. Separation was carried out on a cosmosil C18 column, mobile phase composition was methanol: distilled water (60:40, v/v) at flow rate of 1 mL/min. Detection was carried out at 215 nm using a photodiode array detector (PDA). Commercial Ayurvedic formulations such as Caturjata Churna, Lavangadi Vati, Jatiphaladi Churna, Sitopaladi Churna and clove oil were further subjected to RP-HPLC for estimation of eugenol. The RP-HPLC method was validated as per ICH guidelines. The LOD and LOQ level were found to be 25.00 ng/mL and 50.00 ng/mL, respectively. Detector response was found to be linear from 50.00 ng/mL to 50,000.00 ng/mL. The method was found to be simple, accurate, reproducible and rugged. This method was used to estimate the content of eugenol in commercial formulations and the method is found suitable for quality assurance and marker based standardization of ayurvedic formulations containing eugenol.