Over the course of agricultural and plant pathology history, the swift and accurate detection of plant-associated fungi has undergone a remarkable evolution, significantly impacting disease management strategies. Notably, the adoption of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) stands as a watershed moment in this domain. Initially employed in clinical microbiology, MALDI-TOF MS has emerged as a game-changing tool for identifying plant pathogenic fungi. Through its ability to generate precise spectral fingerprints based on unique protein profiles, this technology has enabled the rapid and reliable detection of fungal strains. Its successful integration into plant pathology has revolutionized fungal identification, empowering practitioners to swiftly diagnose diseases, implement timely intervention measures, and effectively safeguard plant health, thereby contributing significantly to optimizing crop yield and ensuring agricultural sustainability. The current chapter outlines a method for identifying fungal species associated with plants using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Plant-associated fungi play crucial roles in agriculture, forestry, and ecology, making their accurate and efficient detection imperative. MALDI-TOF MS presents an efficient method to identify fungal pathogens, delivering swift outcomes with minimal need for sample preparation, thus enabling high-throughput analysis.