Cross-contamination has been a major concern for both the practitioner and the patient in recent years. Various sterilization equipment’s throw the challenge to choose the best available in the market. Hence the present study was conducted to assess the efficacy of UV disinfection chamber.The present study was an in vitro study conducted using contaminated dental instruments. Autoclaved dental Instruments were used to obtain the swab from carious lesion. These specimens were then inoculated on blood agar and incubated at 37oC for 24 hrs and colony forming units were determined. Thereafter, these instruments were kept in Ultraviolet (UV) Chamber for 5 min and again they were inoculated on blood agar to determine colony forming units. Same experiment was performed for keeping the instruments in UV cabinet for different time periods i.e. 10 min, 15 min, 20 min, 25 min, 30 min, 45 min and 60 min. and no. of colony forming units (CFUs) were again determined before and after keeping the instruments in UV cabinet . The values were subjected to descriptive statistical analysis. UV Disinfection Chamber is most effective when instruments are kept in UV chamber for a period of 45-60 min. Thus UV Disinfection chambers can be used in dental offices as well as dental outreach program as an effective tool for disinfection of instruments.