Proteins derived from plant resources such as oilseed meals, canola and sunflower, are considered a viable alternative to animal proteins for food consumption. This work presents a rational methodology, using high throughput experimentation (HTE), for the separation of cruciferin and napin, the two major proteins of canola meal, by chromatography. Eight different mixed mode and ion exchange resins were evaluated at different conditions with the aim of capturing napin and identifying adsorption/desorption behavior, ease of desorption and selectivity. POROS 50 HS resulted as the most promising resin. The obtained equilibrium adsorption data for napin and cruciferin was used in a mechanistic chromatography model and compared with experimental results showing a very good agreement. The model was used to identify column operating parameters that lead to >98% yield and purity for both proteins. Subsequently a conceptual downstream processing was proposed.