Targeted metabolomics has been broadly used for metabolite measurement due to its good quantitative linearity and simple metabolite annotation workflow. However, metabolite interference, the phenomenon where one metabolite generates a peak in another metabolite's MRM setting (Q1/Q3) with a close retention time (RT), may lead to inaccurate metabolite annotation and quantification. Besides isomeric metabolites having the same precursor and product ions that may interfere with each other, we found other metabolite interferences as the result of inadequate mass resolution of triple-quadruple mass spectrometry and in-source fragmentation of metabolite ions. Characterizing the targeted metabolomics data using 334 metabolite standards revealed that about 75% of the metabolites generated measurable signals in at least one other metabolite's MRM setting. Different chromatography techniques can resolve 65-85% of these interfering signals among standards. Metabolite interference analysis combined with the manual inspection of cell lysate and serum data suggested that about 10% out of ∼180 annotated metabolites were mis-annotated or mis-quantified. These results highlight that a thorough investigation of metabolite interference is necessary for accurate metabolite measurement in targeted metabolomics.