Transcription factors (TFs) are a family of DNA-binding proteins whose gene regulatory capabilities are of vital importance in defining the molecular state of a cell. Despite their biological significance, our understanding of TF behavior and function is still limited. This is because we have so far mostly relied on gene expression data to approximate TF protein levels given that the latter information has been notoriously difficult to obtain due to the relatively low expression levels of many TFs. However, significant advances in mass spectrometry technologies combined with the development of sensitive methodologies aimed at detecting TFs are now allowing a transition from a predominantly qualitative to a quantitative protein landscape. Such a paradigm shift is expected to unravel dynamic aspects of TF function, potentially linking TF copy number fluctuations in cells with specific regulatory functions. This in turn may provide novel insights into the regulatory mechanisms underlying a wide range of fundamental and disease-related biological processes. In this review, we will present the latest advances in mass spectrometry-based TF proteomics and describe novel strategies tailored around the quantification of this important family of DNA-binding proteins.