This study developed and validated a method for the extraction and determination of 11 phenolic acids in rat plasma, urine, and liver by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). A system suitability test (instrumental linearity, area, and retention time precision) was performed and recovery, intraday and between-day precisions, detection limits (LOD), and quantification limits (LOQ) were determined for all compounds in each biological matrix. Recoveries varied between 88 and 117% in plasma, between 87 and 102% in urine, and between 38 and 100% in liver. Precision was higher than 13.7% intraday and 14.0% interday in all matrices, at three concentration levels. To demonstrate the applicability, the method was used to estimate the concentrations of phenolic acids in samples from animals that received 5-caffeoylquinic acid (5-CQA) by gavage. The excellent validation results and the applicability of the method to real samples confirmed the suitability for studies on absorption, bioavailability, and pharmacokinetics of phenolic acids derived from foods rich in phenolic compounds.