Modern high-throughput and high-content biological research is performed with advanced instrumentation and complex and time-consuming protocols, which, as a whole, pose a challenge for routine implementation in a research laboratory. In support of a "bioanalytical toolbox" with potential utility for exploring cellular functions mediated via protein phosphorylation-a post-translational modification (PTM) with essential regulatory roles in a variety of cellular processes-in this work, we describe the development of a simple, integrated microfluidic chip that can perform targeted, quantitative analysis of phosphopeptides involved in cancer-relevant signaling pathways. The microfluidic device comprises microreactors packed with C18 and TiO2 particles for on-chip solid phase extraction (SPE) and phosphopeptide enrichment, and an ESI interface for facilitating multiple reaction monitoring (MRM)-mass spectrometry (MS) detection. The chips are demonstrated for the detection of three phosphopeptides involved in ERBB2/MAPK signaling pathways, selected from the outcome of a proteomic study involving EGF stimulation of SKBR3/HER2+ breast cancer cells. The data demonstrate that the proposed microfluidic strategy can be used for the MS quantification of phosphopeptides in the low nM range from cell lysates without any prior sample pretreatment, fractionation or bioaffinity enrichment, and is generally applicable to the analysis of any phosphopeptide targets.