Development of the vertebrate hair bundle is a precisely orchestrated event that culminates in production of a tightly ordered arrangement of actin-rich stereocilia and a single axonemal kinocilium. To understand how the protein composition of the bundle changes during development, we isolated bundles from young (postnatal days P4-P6) and mature (P21-P25) mouse utricles using the twist-off method, then characterized their constituent proteins using liquid-chromatography tandem mass spectrometry with data-dependent acquisition. Using label-free quantitation, we measured relative abundances of proteins in both bundles and in the whole utricular epithelium; comparison of protein abundance between the two fractions allows calculation of enrichment in bundles. These data will be useful for examining the proteins present in mammalian vestibular bundles and how they change over development.