The last decade has brought rapid advances in our knowledge of the human genome, as well as increasingly sophisticated methods to analyze how each of approximately 30,000 genes within it contribute to cellular, tissue, and organismal function. Here, we review this technological revolution in the context of Polo-like kinase 1 (Plk1), which has emerged as a central regulator of multiple processes fundamental to cell division. In particular, we highlight similarities and differences when Plk1 function is probed through various methods, including novel chemical inhibitors, and how our understanding of Plk1's role in mitosis and cell division has been enhanced as a consequence.