The apoplastic fluid moving in the extracellular space external to the plasma membrane provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Here, we describe an optimized vacuum-infiltration-centrifugation method to extract soluble proteins from apoplastic fluid of grapevine leaves. This optimized method allows recovering of the grapevine apoplastic soluble proteins suitable for mono- and bi-dimensional gel electrophoresis for further proteomic analysis in order to elucidate their physiological functions.