This study represents a systematic evaluation of protocols for protein extraction and cleanup for fruit proteomic analysis. Procedures were optimized using pooled lyophilized banana fruit pulp, which is known to be particularly tricky due to high concentrations of soluble polysaccharides, phenolics, and other substances that interfere with protein extraction and purification. A total of 18 combinations of three protein extraction procedures (SDS-based, Triton X-100-based, and phenol-based), three protein precipitating agents (ammonium acetate/methanol, TCA/acetone, and acetone), and two resolubilization buffers (classical Rabilloud and the so-called R2D2) were compared for total protein yields and efficiency of recovery. The results demonstrate that while losses in total recovered protein are unavoidable, the degree of these losses depends on the method combinations used. Combinations based on buffer-saturated phenol always gave the highest yields, and overall recovery and purity was highest when acetone was combined with the R2D2 buffer for protein purification and concentration. Comparative 2D-PAGE analysis confirmed that this method combination produced high-quality and reproducible gels and the largest numbers of spots per gel. The usefulness of this methodology was demonstrated on ripe fruits from several other species and shown to give excellent results.