Mycoplasma contamination is well known serious microbial contamination that encountered during the production of biopharmaceutical products produced from cell culture. Contamination to this biopharmaceuticals represents potential risk for the health of patients. To minimize this risk monitoring of mycoplasma contamination is preferred during the manufacturing process in cell culture substrates. The current methods recommended by United States Pharmacopoeia, European Pharmacopoeia, British Pharmacopoeia and Indian Pharmacopoeia for mycoplasma testing of biologics involve cell culture methods and indicator cell culture method. The conventional methods require long time period assays (minimum 28 days) and skill for interpretation of the results which limits the use of these assays. The use of Nucleic Acid Amplification Test (NAT) technique provides advantages over conventional microbiological methods in terms of analytical output, sensitivity and turnover time. The present review provide the overview about the conventional technique used, and their limitations, use of nucleic acid based method in mycoplasma testing of biologics and cell substrates. This review also provide the information about current regulatory requirements as prescribed by US-FDA, European Medicines Agency (EMA), World Health Organization (WHO) and guidelines for mycoplasma testing in biologicals, worldwide.