Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasmas can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for the presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based method, indirect staining and an agar and broth culture. This protocol describes these three tests for detecting Mycoplasma, which take from 1 d to 3-4 weeks, and such tests should be an obligatory component of quality control in every tissue culture laboratory.