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The rapid identification and quantitation of alkaloids produced by <i>Epichlo&#235;</i> endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for <i>Epichlo&#235;</i>-infected perennial ryegrass (<i>Lolium perenne</i> L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography&#8722;mass spectrometry (LC&#8722;MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC&#8722;MS (for peramine). This work provides the first highly sensitive quantitative LC&#8722;MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.