This study presents an improved protocol for studying the plant cell wall proteome, comparing three extraction methods for cell wall proteins (CWPs) from alfalfa stems. The three-step protocol, which uses sequential buffers containing CaCl₂, EGTA, and LiCl, was found to identify the highest number of CWPs (242 NCBInr identifiers) and the lowest percentage of non-CWPs (about 30%). However, the three protocols are complementary rather than substitutive, as 43% of the identified proteins were specific to one protocol. The three-step protocol was selected for further proteomic characterization using 2D-gel electrophoresis, which showed that 75% of the identified proteins were fraction-specific and 72.7% were predicted to belong to the cell wall compartment. Although less sensitive than LC-MS/MS in detecting low-abundant proteins, gel-based approaches are valuable for differentiating and quantifying protein isoforms and modified proteins. The study highlights the importance of combining different extraction and analytical techniques to understand the dynamics of the plant cell wall proteome. Data are available via ProteomeXchange with identifier PXD001927. Keywords: proteomics, cell wall, plant, glycosylation, EGTA.This study presents an improved protocol for studying the plant cell wall proteome, comparing three extraction methods for cell wall proteins (CWPs) from alfalfa stems. The three-step protocol, which uses sequential buffers containing CaCl₂, EGTA, and LiCl, was found to identify the highest number of CWPs (242 NCBInr identifiers) and the lowest percentage of non-CWPs (about 30%). However, the three protocols are complementary rather than substitutive, as 43% of the identified proteins were specific to one protocol. The three-step protocol was selected for further proteomic characterization using 2D-gel electrophoresis, which showed that 75% of the identified proteins were fraction-specific and 72.7% were predicted to belong to the cell wall compartment. Although less sensitive than LC-MS/MS in detecting low-abundant proteins, gel-based approaches are valuable for differentiating and quantifying protein isoforms and modified proteins. The study highlights the importance of combining different extraction and analytical techniques to understand the dynamics of the plant cell wall proteome. Data are available via ProteomeXchange with identifier PXD001927. Keywords: proteomics, cell wall, plant, glycosylation, EGTA.