Data-dependent acquisition (DDA) mode is widely applied in shotgun proteomics. However, restricted by the scanning speed of mass spectrometers, it"s difficult for DDA to directly detect peptides with low abundance. Herein, we introduce isoSTAR as a real-time isotopic signature targeted profiling platform, which identifies target peptides by their unique isotopic signatures during the full-MS scanning stage and schedule targeted MS/MS scans in no time. The isoSTAR showed dramatical improvement of sensitivity and identification of targets compared to traditional acquisition methods, which significantly enhanced the discovery of novel PTMs and the profiling of biomacromolecule interactions in practice. We envision that isoSTAR can become a convenient and powerful tool in shot-gun proteomics, providing enhanced and accurate peptide profiling.