Rust diseases are a major limitation to grain production in Canada and have caused devastating yield losses worldwide [1]. The identification and characterization of pathogenic fungi responsible for rust diseases are important for implementing effective disease management practices. Modern DNA-based methods of identification, such as quantitative polymerase chain reaction (PCR) and sequencingSequencing, are time-consuming, costly, and require well-trained laboratory personnel. Here, we describe a protocol to rapidly identify rust samples to the species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MSMatrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)). Biochemical profiles generated by MALDI-TOF MSMatrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) are used for identification. The method is commonly used in clinical settings and can accurately identify hundreds of microbes in minutes at a low cost. The MALDI-TOF MSMatrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) method described here is simple and can assess samples of rust spores and spore-mats originating from different cereal hosts and identify them to the species level with high accuracy, high throughput, and low cost. The method is not currently able to accurately distinguish among races, though this may be possible with additional research and sample fractionation.