In the realm of global agriculture, India stands as the second-largest crop producer but grapples with significant challenges like pest outbreaks and climate change. Identifying pathogens, including diverse plant-associated bacteria, is crucial for the effective treatment against pathogens. Accurate identification of these bacterial communities has historically been achieved through cultivation-based techniques and molecular methods such as PCR and 16S rRNA gene sequencing. With the evolving demands of agricultural and ecological research, a paradigm shift toward rapid, reliable, and high-throughput methodologies has been found. Recent advancements in mass spectrometry, specifically MALDI-TOF MS, have provided an opportunity to swiftly characterize bacterial species based on their unique proteomic profiles. This chapter introduces a comprehensive protocol tailored for the detection of plant-associated bacteria using MALDI-TOF MS beginning with sample collection, preparation, analysis, and data interpretation. This chapter aims to streamline the identification process, ensuring accurate results and fostering a deeper understanding.