Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has become an increasingly popular method for bacterial identification in recent years. Contagious Equine Metritis is a venereal disease of equids of international significance caused by the gram negative organism, Taylorella equigenitalis. The prescribed test is by bacteriological culture, however, due to the slow-growing and fastidious nature of the bacteria, culture requires specialized media and extended culture time. In addition, differentiation of T. equigenitalis and the closely related Taylorella asinigenitalis from each other and from common contaminants of the equine genital tract is difficult and has required advanced diagnostics with specialized reagents. This study evaluates the use of MALDI-TOF identification for detecting and differentiating individual colonies of T. equigenitalis and T. asinigenitalis directly from culture plates, significantly decreasing the time and cost of diagnosis. The study compared the effects of varying extraction methods, types of culture media and day of culture on the ability to identify these species. This method represents an inexpensive, rapid, and accurate new diagnostic tool for Contagious Equine Metritis diagnosis.