Signal transducers and activators of transcription (STATs) are implicated in physiological cellular processes, stress-related responses, and malignancies. This presentation provides an overview on STATs, focusing on STAT5, and describes an in vivo biotinylation methodology applied in mammalian cells for identification of STAT5-containing protein complexes and STAT5 target genes. Our results show that in vivo biotinylation can be used efficiently to elucidate the complex STAT5-dependent transcriptional mechanisms.