Cotton (Gossypium hirsutum L.) is a highly significant crop because of its quality for textile industry of the world. With the advent of genetic engineering, cotton has gone through several genetic improvements that encode tolerance against biotic and abiotic stresses. The efficient seed sterilization method is essential for successful genetic improvement in cotton.  The present study was conducted to compare different sterilizing agents employed for in vitro culture of cotton to find out an efficient, reproducible, cost effective and genotype independent in vitro seed sterilization procedure of cotton. The two cultivated cotton cultivars i.e. GSN-12 and Coker-312 were used for these studies that were further transformed with insecticidal genes under different promoters. The different sterilizing agents used (in separate or combination) were ethanol, commercial bleach (NaOCl), mercuric chloride (HgCl2), Sodium Dodecyle (SDS), hydrogen peroxide (H2O2). The surface sterilized seeds of both cultivars were cultured on MS medium and data was recorded for contamination free seeds and germination ratio. The results showed that 5% n-Hexane in combination with 2% of H2O2 are the best sterilizing agents for both cultivars when compared to the efficacy of other chemicals. Seeds treated with n-hexane and hydrogen peroxide had minimum microbial contamination with improved germination rate. The sterilizing method was successfully used for surface sterilization of other cotton cultivars in our project. According to our knowledge, this is the first report of using n-hexane in combination with H2O2 for surface sterilization of cotton seed for in vitro culture studies.