The vast functionality of thousands of proteins stems from their diverse chemical make-up brought about by the linkage of amino acids through amide bonds. In food systems, proteins are known to stabilize protein gelation, the air-oil interface during emulsion, and the air-water interface during foaming. The aim of this study was to determine protein concentration, free amino acid content and protein size of the Opuntia cladode mucilage. A further aim was to compare the protein concentration of Opuntia cladode mucilage to that of commercial hydrocolloids currently used in the food industry to estimate their performance in a food system. Mucilage was extracted and freeze-dried from 42 cactus pear cultivars obtained from an experimental orchard on the University of the Free State campus. The nitrogen and protein concentration, free amino acid content and protein size were determined by thermal combustion (Leco) and Bradford assay, Cadmium-ninhydrin method, and SDS-PAGE, respectively. The average protein concentration obtained by the Leco method was 5.44% and 3.61% by the Bradford assay. Vryheid had the highest protein concentration for both Leco (10.15%) and Bradford (8.25%) assays, respectively. Of the five cultivars selected for further characterization, Gymno-Carpo (30 kDa) had the largest protein size. The Opuntia mucilage showed to contain free amino acids. Opuntia mucilage had protein concentrations similar to that of casein, xanthan, and guar gum.