Reversible phosphorylation of proteins controlled by kinases and phosphatases is one of the most ubiquitous post-translational modifications, and regulates a variety of cell functions through cellular signal transduction pathways. These signals are involved in various diseases such as cancer and rheumatism, and often cause the disease itself or drive the progression. Quantitative phosphoproteomics based on liquid chromatography-tandem mass spectrometry combined with phosphopeptide enrichment and stable isotope labeling allows profiling thousands of phosphorylation sites on human proteins and has been applied to monitoring cellular phosphorylation dynamics induced by various growth factors, hormones and other perturbations including kinase inhibitors. Here, we employed these technologies to quantify the temporal response of phosphorylation dynamics in SKBR3 breast cancer cells to lapatinib, a kinase inhibitor for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 μM lapatinib. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.