Diundecyl phthalate (DUP) is a high production volume chemical used as a plasticizer in polyvinyl chloride and other plastics. Specific biomarkers of DUP would be useful for human exposure assessment. To identify such biomarkers, we investigated the in vitro metabolism of DUP with human liver microsomes using online solid phase extraction coupled to HPLC-mass spectrometry. Using high resolution mass spectrometry, we conclusively confirmed the structures of four DUP specific metabolites: mono-undecyl phthalate (MUP), mono-hydroxyundecyl phthalate (MHUP), mono-oxoundecyl phthalate (MOUP), and mono-carboxydecyl phthalate (MCDP). We also used high resolution mass spectrometry to isolate MCDP and MHUP from co-eluting isobaric metabolites of diisononyl phthalate (i.e., mono-carboxyisononyl phthalate) and diisododecyl phthalate (i.e., monohydroxyisododecyl phthalate), respectively, that could not be separated with low resolution tandem mass spectrometry. To evaluate the potential usefulness of the newly identified DUP metabolites as exposure biomarkers, we analyzed 36 human urine samples by high resolution mass spectrometry. We detected MHUP and MCDP in > 83% of the samples; median concentrations were 0.21 ng/mL and 0.36 ng/mL, respectively. MOUP was detected only in 14% of the samples analyzed, and MUP was not detected. All three metabolites eluted as peak clusters likely because of the presence of multiple oxidation sites and multiple isomers in DUP technical mixtures. Taken together, these findings suggest that with the appropriate mass spectrometry quantification techniques, MHUP and MCDP may serve as suitable biomarkers for assessing background exposure to DUP.