Polyphenols, prevalent in plants and fungi, are investigated intensively in nutritional and clinical settings because of their beneficial bioactive properties. Due to their complexity, analysis with untargeted approaches is favorable, which typically use high-resolution mass spectrometry (HRMS) rather than low-resolution mass spectrometry (LRMS). Here, the advantages of HRMS were evaluated by thoroughly testing untargeted techniques and available online resources. By applying data-dependent acquisition on real-life urine samples, 27 features were annotated with spectral libraries, 88 with in silico fragmentation, and 113 by MS1 matching with PhytoHub, an online database containing >2000 polyphenols. Moreover, other exogenous and endogenous molecules were screened to measure chemical exposure and potential metabolic effects using the Exposome-Explorer database, further annotating 144 features. Additional polyphenol-related features were explored using various non-targeted analysis techniques including MassQL for glucuronide and sulfate neutral losses, and MetaboAnalyst for statistical analysis. As HRMS typically suffers a sensitivity loss compared to state-of-the-art LRMS used in targeted workflows, the gap between the two instrumental approaches was quantified in three spiked human matrices (urine, serum, plasma) as well as real-life urine samples. Both instruments showed feasible sensitivity, with median limits of detection in the spiked samples being 10-18 ng/mL for HRMS and 4.8-5.8 ng/mL for LRMS. The results demonstrate that, despite its intrinsic limitations, HRMS can readily be used for comprehensively investigating human polyphenol exposure. In the future, this work is expected to allow for linking human health effects with exposure patterns and toxicological mixture effects with other xenobiotics.