Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the 13Ca, 13Cb, 13C&#8242;, 15N, 1Ha and 1HN NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, prior to the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the PDB, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues yielded full atom models that have 0.7-1.8 root-mean-square deviations for the backbone atoms relative to the experimentally determined X-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular weights up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination. Application to the homodimeric protein TolR, supplemented by small angle Xray scattering data, was able to generate a high-resolution structure that was virtually indistinguishable from the structure determined in a time-consuming manner using the conventional NMR strategy.