The multicomponent analysis of human breast milk (BM) by metabolic profiling is a new area of study applied to determining milk composition, and is capable of associating BM composition with maternal characteristics, and subsequent infant health outcomes. A multi-platform approach combining high-performance as well as ultra-performance liquid-chromatography (HPLC-MS and UPLC-MS), gas chromatography (GC-MS), capillary electrophoresis (CE-MS) coupled to mass spectrometry and (1) H NMR spectroscopy was used to comprehensively characterize metabolic profiles from seventy BM samples. A total of 710 metabolites spanning multiple molecular classes were defined. The utility of the individual and combined analytical platforms was explored in relation to numbers of metabolites identified, as well as the reproducibility of the methods. The greatest number of metabolites were identified by the single phase HPLC-MS method, whilst CE-MS uniquely profiled amino acids in detail and NMR was the most reproducible, whereas GC-MS targeted volatile compounds and short chain fatty acids. Dynamic changes in BM composition were characterized over the first 3 months of lactation. Metabolites identified as altering in abundance over lactation included fucose, di- and triacylglycerols and short chain fatty acids, known to be important for infant immunological, neurological and gastrointestinal development, as well as being an important source of energy. This extensive metabolic coverage of the dynamic BM metabolome provides a baseline for investigating the impact of maternal characteristics, as well as establishing the impact of environmental and dietary factors on the composition of BM, with a focus on the downstream health consequences this may have for infants. This article is protected by copyright. All rights reserved.