Both of these approaches have been applied to investigate polyphenols, however, they were mainly applied on foodstuffs rather than human matrices , especially for untargeted workflows . Thus, the aim of this study was to transfer a previously validated method for a vast set of polyphenol standards 17 from a targeted LRMS to an untargeted HRMS workflow. This HRMS workflow was developed using suspect screening and four different non-targeted analysis approaches on urine samples from a pilot study. This allowed for a thorough test of untargeted data analysis techniques, evaluation of available online resources, and exploration of potential limitations. As HRMS is known to be less sensitive than LRMS, this gap was quantified to better characterize the applicability of HRMS on polyphenol analysis in human biofluids. This was achieved by comparing the limits of detection (LODs) of both workflows for 90 polyphenol reference standards from a variety of classes. Finally, both workflows were applied to case study samples to compare their performance at naturally occurring concentrations.