Transcription factors are desirable and under-exploited targets for small molecule direct intervention, but molecular control has been difficult to achieve. For the master hematopoietic transcription factor PU.1, in solution and in cellulo methodologies for scoring ligands with putative functional activities were developed. Using fluorescence polarization and cellular reporter assays, the modulation of PU.1/DNA binding by peptide-based ligands were characterized. Several peptides were found to enhance PU.1 affinity for a fluorescently labeled DNA probe by a factor of ~2 relative to vehicle, while others inhibited binding by ~20-fold in equilibrium dissociation constant (KD) relative to vehicle. In THP-1 cells (a human monocytic cell line that natively expresses PU.1), fluorescent reporters measured ~1.2-fold enhancement in phenotypic output of PU.1 transactivation in the presence of enhancement peptides and ~1.1-fold detriment in the presence of inhibitors. These results establish methodology to characterize pharmacologic interactions of the transcription factor PU.1.