This study compares three protocols for extracting cell wall proteins from alfalfa stems: a three-step procedure using CaCl2, EGTA, and LiCl buffers, and two previously published methods. The three-step protocol identified the highest number of cell wall proteins (242 NCBI nr identifiers) with the lowest percentage of non-cell wall proteins (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 analysis using 2D-gel electrophoresis, which revealed that 75% of the identified proteins were fraction-specific and 72.7% were predicted to be located in the cell wall. While gel-based approaches are less sensitive than LC-MS/MS in detecting low-abundant proteins, they are valuable for differentiating and quantifying protein isoforms and post-translational modifications. The study highlights the complementary nature of different extraction and analytical techniques in studying the plant cell wall proteome.This study compares three protocols for extracting cell wall proteins from alfalfa stems: a three-step procedure using CaCl2, EGTA, and LiCl buffers, and two previously published methods. The three-step protocol identified the highest number of cell wall proteins (242 NCBI nr identifiers) with the lowest percentage of non-cell wall proteins (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 analysis using 2D-gel electrophoresis, which revealed that 75% of the identified proteins were fraction-specific and 72.7% were predicted to be located in the cell wall. While gel-based approaches are less sensitive than LC-MS/MS in detecting low-abundant proteins, they are valuable for differentiating and quantifying protein isoforms and post-translational modifications. The study highlights the complementary nature of different extraction and analytical techniques in studying the plant cell wall proteome.