Analysis of multiple protein–protein interactions using microarray technology remains challenging, and site-specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein–DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt–DNA hybrid molecules described here are used for DNA-directed immobilization on glass- and silica-based microarrays. Methods for the detection of protein–DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP- and GTP-bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt-effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.