Differential gene expression is tightly controlled by transcription factors (TFs), which bind close to target genes and interact together to activate and coregulate transcription. Bioinformatics analysis of published genome-wide gene expression data has allowed the development of comprehensive models of TFs likely to be active in particular tissues (signature TFs); however, the predicted activities of many of the TFs have not been experimentally confirmed. Here, we describe methods for the parallel analysis of the activities of more than 200 transcription factor proteins, using an advanced oligonucleotide array-based transcription factor assay (OATFA) platform, to assay TF activities in mice. The system uses a PCR-based system to translate cellular levels of target DNA-TF complex into a dye-tagged DNA signal, which is read by the developed microarray. The PCR step introduces semiquantitative amplification of the represented TF binding sequences. Experimental OATFA findings can identify many TF activities, which bioinformatics profiling does not predict. Newly identified TF activities can be confirmed by antibody-ELISA against active TFs. The PCR-based OATFA microarray analysis is a comprehensive method that can be used to reveal transcriptional systems and pathways which may function in different mammalian tissues and cells.