The understanding of complex biological systems is still hampered by limited knowledge of biologically relevant quaternary protein structures. Here， we demonstrate quaternary structure determination in biological samples using a combination of chemical cross-linking， high-resolution mass spectrometry and high-accuracy protein structure modeling. This approach， termed targeted cross-linking mass spectrometry (TX-MS)， relies on computational structural models to score sets of targeted cross-linked peptide signals acquired using a combination of mass spectrometry acquisition techniques. We demonstrate the utility of TX-MS by creating a high-resolution quaternary model of a 1.8 MDa protein complex composed of a pathogen surface protein and ten human plasma proteins. The model is based on a dense network of cross-link distance constraints obtained directly in a mixture of human plasma and live bacteria. These results demonstrate that TX-MS can increase the applicability of flexible backbone docking algorithms to large protein complexes by providing rich cross-link distance information from complex biological samples.