A plethora of gene families encoding known or predicted secreted regulatory peptides occur in diverse plants. Characterised peptides include CLE, CEP, RGF, IDA, PSY, PIP and CIF gene family members, which collectively influence several developmental and defence-related responses. These peptides are secreted at low nM concentrations and act as local or long-distance signals. The peptides are encoded within short open-reading-frames and broadly share a common structure: an N-terminal secretion signal, a variable region of unknown function, one or more conserved peptide domains ranging from 5-20 amino acids (depending upon the family). The mature peptides are post-translationally modified. We devised an approach to efficiently elucidate the structures and post-translational modifications of diverse peptide hormones in plant secreted fluids to simultaneously identify and characterise several potential long-distance peptide hormones. We combined an enrichment procedure with mass spectrometry and bioinformatics to comprehensively analyse the secreted peptidome of Medicago truncatula root cultures and soybean xylem sap-a fluid that moves to the shoot carrying root-to-shoot signalling molecules (1). Multiple spectra corresponding to four peptide hormone families (CEPs, CLEs, XAPs and CIFs) were found to be secreted from Medicago root cultures and present in soybean xylem sap, suggesting that these peptides are long-distance signals. Common PTMs identified included proline hydroxylation and glycosylation and tyrosine sulfation. Several CEP and XAP peptides possessed C- and sometimes N-terminal amino acids that extended beyond the predicted conserved peptide domain borders and their diverse structures suggested roles for endo- and exoproteases in peptide maturation (1). Several peptides were chemically synthesized to probe the effect of post-translational modifications and variations in length on function. Since root cultures can be made on many plants, they may represent an ideal source to simultaneously and efficiently identify diverse peptide hormones in vivo that may play long-distance signalling roles.