The H-point curve isolation method (HPCIM) for thedetection of unknown interferences in chromatography isproposed. The method allows one to estimate the UV−vis spectra of interfering species in a sample as well as totest the purity of the chromatographic peaks. Besides thedetection of the unknown interferences in a sample, thismethod allows one to calculate the concentration of ananalyte in the presence of unknown compounds. Toillustrate the reliability of the proposed method, samplesof diuretics and amphetamines have been analyzed bynormal- and reversed-phase high-performance chromatography.