Conjugation reactions are of critical significance in human metabolism. Identification of these conjugated metabolites is still challenging. Here, we propose a strategy, post-deconvolution MS/MS spectra extraction with data-independent acquisition (PDMS2E-DIA), to comprehensively profile the glucuronide-conjugated metabolome. PDMS2E-DIA enables the identification of conjugated and unconjugated metabolite pairs through neutral loss filtering combined with a significant change in abundance after the deconjugation reaction. Purified DIA MS/MS spectra were constructed by extracting MS/MS fragments shared between spectra derived from conjugated and unconjugated metabolites. The feasibility of this approach was first demonstrated by the identification of two glucuronide-conjugated metabolite standards spiked in urine samples. For human urine samples, 479 features were structurally annotated as potential glucuronide-conjugated metabolites, resulting in the identification of 211 metabolites. Fragment peaks derived from interferents were found to be removed by PDMS2E-DIA, which increased about 6 times the number of identified urine metabolites compared with those calculated from raw DIA deconvoluted MS/MS spectra. This approach was found to have great potential for identifying glucuronide-conjugated metabolites, as well as other kinds of chemical conjugations.