Quantitative biology requires quantitative data. No high-throughput technologies exist capable of obtaining several hundred independent kinetic binding measurements in a single experiment. We present an integrated microfluidic device (k-MITOMI) for the simultaneous kinetic characterization of 768 biomolecular interactions. We applied k-MITOMI to the kinetic analysis of transcription factor (TF)—DNA interactions, measuring the detailed kinetic landscapes of the mouse TF Zif268, and the yeast TFs Tye7p, Yox1p, and Tbf1p. We demonstrated the integrated nature of k-MITOMI by expressing, purifying, and characterizing 27 additional yeast transcription factors in parallel on a single device. Overall, we obtained 2,388 association and dissociation curves of 223 unique molecular interactions with equilibrium dissociation constants ranging from 2 × 10 -6 M to 2 × 10 -9 M, and dissociation rate constants of approximately 6 s -1 to 8.5 × 10 -3 s -1 . Association rate constants were uniform across 3 TF families, ranging from 3.7 × 10 6 M -1 s -1 to 9.6 × 10 7 M -1 s -1 , and are well below the diffusion limit. We expect that k-MITOMI will contribute to our quantitative understanding of biological systems and accelerate the development and characterization of engineered systems.