Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.