We describe a method for the simultaneous extraction of proteins and nucleic acids from Medicago truncatula tissues. Using a modified TRIzol reagent method, we developed a simple and an effective way to simultaneously extract proteins and nucleic acids from a single sample. We verified that this method does not affect the quality or quantitation of the isolated DNA and RNA. Furthermore, we used 2-DE to compare M. truncatula leaf, stem, and root samples processed using this new method with two commonly used methods: phenol extraction/methanol-ammonium acetate precipitation and trichloroacetic acid/acetone precipitation. The results showed that our method was superior to the other methods, based on 2-DE patterns. We also demonstrated that our protocol is compatible with proteomic analysis, as 10 out of 14 selected proteins isolated by the method were identified by MALDI-TOF-MS/MS. The protocol described can be used with sample preparation protocols for proteomic, transcriptomic, and genomic studies.