An efficient protein extraction method is crucial to ensure successful separation by two‐dimensional electrophoresis (2‐DE) for recalcitrant plant species, in particular for grapevine (Vitis vinifera L.). Trichloroacetic acid‐acetone (TCA‐acetone) and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar ‘Razegui’. The phenol‐based protocol proved to give a higher protein yield, a greater spot resolution, and a minimal streaking on 2‐DE gels for both leaf and root tissues compared with the TCA‐based protocol. Furthermore, the highest numbers of detected proteins on 2‐DE gels were observed using the phenol extraction from leaves and roots as compared with TCA‐acetone extraction.