The stability of vaccines has a major impact on the success of immunization programmes worldwide. As part of its efforts to assure vaccine quality, ICH harmonized guideline for stability testing of new drug substances and product Q1A (R2) on 6th February 2003 has acknowledged the importance of clearly defining the stability characteristics of a vaccine and emphasizes the role of national regulatory authorities in overall vaccine evaluation. Therefore, this study was initiated with the aim of investigating the real-time stability of lyophilized Sabin formulated bivalent Oral Polio Vaccine (sbOPV) at room temperature. The stability of the lyophilized sbOPV was investigated through various quality attributes at room temperature for two consecutive years in our laboratory, such as physiochemical quality attributes (Colour, Appearance, Moisture content, pH and analysis of Magnesium Chloride) and biological including microbiological quality attributes (potency, identity, sterility, and kanamycin antibiotic activity). The results of the study revealed that lyophilized sbOPV is equally potent in comparison to currently used liquid sbOPV and also found stable at room temperature for two years, and also suggested that there is no need to maintain the cold chain system during transportation, and storage of lyophilized sbOPV.