Multiple reaction monitoring (MRM) of peptides employs tandem mass spectrometry to quantify selected proteins of interest, such as those previously identified in differential studies. Using this technique, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. The design of transitions is critical for the success of MRM experiments, but predicting signal intensity of peptides and fragmentation patterns ab initio is challenging given existing methods. The tool presented here, MRMaid (pronounced ‘mermaid’) offers a novel alternative for rapid design of MRM transitions for the proteomics researcher. The program uses a combination of knowledge of the properties of optimal MRM transitions, taken from expert practitioners and literature, with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. The tool also predicts retention time using a published model, allowing ordering of transition candidates. By exploiting available knowledge and resources to generate the most reliable transitions, this approach negates the need for theoretical prediction of fragmentation and the need to undertake prior ‘discovery’ MS studies. MRMaid is a modular tool built around the Genome Annotating Proteomic Pipeline (GAPP) framework, providing a web-based solution with both descriptive and graphical visualizations of transitions. Predicted transition candidates are ranked based on a novel transition scoring system and users may filter the results by selecting optional stringency criteria, such as omitting frequently modified residues, constraining the length of peptides or omitting missed cleavages. Comparison with published transitions showed that MRMaid successfully predicted the peptide and product ion pairs in the majority of cases, with appropriate retention time estimates. As the data content of the GAPP repository increases, MRMaid’s coverage and reliability is set to increase further. MRMaid is freely available over the internet as an executable web-based service at http://www.mrmaid.info.