Cu(I) active sites in metalloproteins are involved in O-2 activation, but their O-2 reactivity is difficult to study due to the Cu(I) d(10) closed shell which precludes the use of conventional spectroscopic methods. K beta X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of K beta XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double-ionization (DI) states are identified using varying incident X-ray photon energies, and a reasonable method to correct the data to remove DI contributions is presented. K beta XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy K beta(2,5) emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy K beta(2,5) emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d-p mixing. A K beta XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GO(pre)) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy K beta(2,5) emission feature in the Cu(I)-GO(pre) data has information about the MO containing mostly Cu 3d(x2-y2) character that is the frontier molecular orbital (FMO) for O-2 activation, which shows the potential of K beta XES in probing the Cu(I) FMO associated with small-molecule activation in metalloproteins.