This project focuses on the proteomic analysis of apoplastic fluids (APFs) extracted from cotton (Gossypium hirsutum) leaves using an optimized infiltration-centrifugation protocol. The APFs, obtained through a meticulous process involving vacuum infiltration, drying, and two-step centrifugation, are free from cellular contamination and are important for in-depth protein profiling to understand leaf physiological functions and responses to environmental stimuli.