Medicago truncatula has become the focus of systems biology research for improved legume crop breeding. In plant systems biology, several comparative studies have been carried out using liquid chromatography shotgun mass spectrometry (LC-MS/MS) and database-dependent protein identification analyses in combination with the spectral count for relative quantification. In order to receive optimal protein identification rates and spectral count quantification, data-dependent tandem mass spectrometry with LC separation of more than 1 h is required. Thus LC-MS/MS analyses time is the bottleneck for high-throughput research of experiments with high sample number.We describe a novel method, called full-scan (FS) selective peptide extraction, that allows for comparative quantification of target peptides combined with a significant reduction in LC-MS analysis time. In future, it will be a useful tool to detect (15)N-labeled selected peptide patterns for the targeted analysis of protein turnover and synthesis. We provide a first reference library of selected target peptides generated for M. truncatula leaf tissue. These peptides are also suitable candidates for selective reaction monitoring approaches.