Transcription factors (TFs) play a central role in regulating gene expression and in providing interconnecting regulatory networks between related pathway elements. Although single TF assays provide some insights into pathway regulation, a method that allows the parallel investigation of all active TFs is highly desired to elucidate the complex inter-regulated cellular mechanisms. We have developed a novel oligonucleotide array-based transcription factor assay platform for genome-wide active TF profiling of Saccharomyces cerevisiae, which can simultaneously analyze the activities of 93 different TFs. The platform has been validated using 28 purified TFs produced in Escherichia coli, cell extracts from yeast strains overexpressing particular TFs, and by detailed control experiments. We then used the platform to examine the activity changes of all yeast TFs during diauxic shift, and results showed, in good agreement with previous studies, that the Sip4 was induced specifically. Other individual TFs required for growth in synthetic complete medium were also identified. Genome-wide analysis of TF activity is extremely useful in investigating complex gene regulatory networks and for the development of systematic understanding of the complexity of genomic functions. These results obtained in this report demonstrate the validity, and for the first time the utility, of this technology for genome-wide investigation of TF activities.