Protein-DNA interactions (PDIs) are critical for many cellular processes. We present here a protocol for the identification of PDIs in vitro using protein microarray technology. The procedure involves double-stranding synthesized DNA oligonucleotides with a fluorescent-labeled primer, binding the labeled double-stranded DNA directly to the protein microarray, and analyzing binding of the resulting PDIs. This approach provides simultaneous identification of PDIs for thousands of proteins, and multiple carefully designed DNA probes can be tested in parallel, which enables a rapid mapping of PDIs on a proteome-wide scale.