Abstract: A qualitative analysis method to identify related substances of rifampicin by correlation of spectra in chromatography was established. Standard chromatographic and spectral data obtained by high-performance liquid chromatography with diode array detection (HPLC-DAD) were developed and used in identification of peaks in samples by calculating correlation coefficients (R). The comparison of spectra was expanded to three-dimensional space. Peaks in chromatography can be identified accurately and rapidly through chromatographic system change. The identification results were validated by LC-MS. The threshold of R was set to 0.99 in order to discriminate the impurities and the minimal amount for identification was 30 ng. Taking full advantage of chromatographic separate efficiency and spectral exclusiveness, the method is accurate and reproducible as a new and effective way to identify multi-components in drugs.