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Annual ryegrass toxicity (ARGT) is a frequently fatal disease that primarily affects livestock in southern and western Australia. The causative agent of the disease is <i>Rathayibacter toxicus</i>, a bacterium that often contaminates the indigenous grasses used for grazing and hay-making. <i>R. toxicus </i>produces a tunicamycin-like toxin under certain conditions, causing extreme hepatic and neurological damage in livestock that consume contaminated grass. Despite the significant toll exacted on infected livestock and its ever-increasing geographical prevalence, little is known about this pathogen. Mass spectrometry (MS) proteomic analysis was performed on bacterial lysates and supernatants under various growth conditions in an effort to elucidate protein expression patterns. This approach provides insight into the basic expression profile of <i>Rathayibacter</i> under benign vs. toxin-producing conditions, as well as facilitating the identification of proteins which may be utilized to generate diagnostic assays, allowing for rapid detection of <i>R. toxicus</i> prior to livestock ingestion. The initial MS analysis of cultured <i>R. toxicus</i> identified 290 expressed proteins, representing the typical biological categories, with the largest number of proteins related to protein synthesis. Bioinformatic analysis identified 9 unique proteins that were viable candidates for antibody production. These results are a significant first step toward a comprehensive evaluation of the <i>R. toxicus</i> proteome.