This work was aimed at optimizing a protein extraction procedure for date palm (Phoenix dactylifera L.) leaves, a highly recalcitrant plant tissue for 2-DE. Five protein extraction protocols based on different protein precipitation agents (TCA/acetone vs. phenol (Ph) methods) and protein resolubilization methods (physical treatments, e.g., sonication, shaking and/or heating) were tested. Ph/SDS extraction with methanol/ammonium acetate precipitation, followed by DOC preincubation and TCA/acetone precipitation and, finally, solubilization by shaking in rehydration solution was found to be the best protein extraction method. We conclude that DOC with TCA/acetone precipitation step eliminates interfering compounds, thus allowing efficient resolubilization of date palm leaf proteins. This method could be appropriate for proteomic studies such as date palm colonization by entomo-pathogenic fungi.