By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the Q(B) site semiquinone (SQ(B)) was determined. The development of such a technique is crucial toward an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQ(B) hydrogen bond network was investigated with Q- (similar to 34 GHz) and X-band (similar to 9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQ(B) g-tensor reference frame provided the principal values and directions of the H-bond proton hyperfine tensors. Three protons were detected, one with an anisotropic tensor component, T = 4.6 MHz, assigned to the histidine N delta H of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQ(B) g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQ(B) used in previous DFT work is in strong agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.