Label-free detection technologies have been widely used to characterize biomolecular interactions without having to label the target molecules. These technologies exhibit considerable potential in facilitating assay development and enabling new integrated readouts. When combined with high-throughput capability, label-free detection may be applied to small molecule screens for drug candidates. Based on the resonant waveguide grating biosensors, a label-free high-throughput detection system, the Epic(®) System, has been applied to monitor molecular interactions. Here we describe a generic label-free assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with the solution-based fluorescence anisotropy assay, our results support that the high-throughput resonant waveguide grating biosensor system has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. Hence, it provides a new generic HTS platform for phospho-detection.