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In recent years, single molecule force spectroscopy has opened unique possibilities to investigate the mechanical properties of single protein molecules. So far, experiments have focused on the mechanical behavior of a protein's tertiary structure. For a large number of proteins, however, multiple folded protein molecules are arranged into a multi-subunit complex. How the quaternary structure of such a complex responds to force is not clear. Here, we present a toolkit to study the mechanical properties of dimeric and trimeric protein complexes by single molecule force spectroscopy. We apply these methods to two different model systems:The dimeric α-crystallin domain is the building block of Hsp 16.5 from Methanococcus janashii. We can directly observe that this dimer dissociates at ∼ 200 pN into two metastable monomeric subunits, which subsequently unfold independently.The two most N-terminal domains of human titin are assembled into an antiparallel complex by telethonin in the Z-disk region of the sarcomere. We show that the Ig domains of titin are stabilized in the presence of telethonin, and that this stabilization is optimized to provide a high level of mechanical strength in the sarcomere. The dissociation force of the titin-telethonin complex exceeds 600 pN, making it one of the highest rupture forces known to date.Single molecule force spectroscopy allows us not only to measure directly the dissociation forces of protein complexes, but also to observe the mechanical hierarchy of the involved building blocks. Building on these results, future experiments will attempt to observe the assembly of protein complexes using single molecule force spectroscopy.