This study was aimed to provide principle of the chemical analyses, antibacterial efficiency test and validation procedures of the most commonly used benzalkonium chloride (BAC) base disinfectant as a biocide. Disinfectant which comprised 20 % BAC concentration was used as a prototype product and active substance was verified with chemical analysis. Additionally, BAC homologs (n-C12 and n-C14) in the product were identified by LC-MS/MS method. The validation results also confirmed the reliability of the chromatographic method. After active substance was confirmed, microbiological efficacy test was carried out against E. coli K12, S. aureus, P. aerugionas and E. hirae. Both bacterial strains were showed more than 5 logaritmic reduction after exposure for 1 min. to test product. The aim of this study was to evaluate the validation of chemical analysis of the test product by LC-MS/MS which is developed chromatographic method. Moreover the present study highlights that the antibacterial efficacy of BAC based disinfectant according to European National (EN) Standard. These results exhibited that chemical and microbiological analyses were significantly important before manufacturing and marketing processes of the biocidal products. This was the first study on biocides analysis which was conducted according to the European standard.