The interest in studying hops and beer prenylflavanoids, isoxanthohumol, xanthohumol, and 8-prenylnaringenin, has increased in recent years due to their biological activity as strong phytoestrogens and potent cancer chemopreventive agents. However, prenylflavanoids behave differently from most polyphenols, since they are unstable at acidic pH. To our knowledge, no published studies to date have considered the degradation of these compounds during analytical processes. In the present work, a new sensitive and specific method based on solid phase extraction and liquid chromatography coupled to electrospray ionization triple quadruple mass spectrometry (LC-ESI-MS/MS) was developed and validated. The new method was optimized to avoid degradation of the selected analytes, isoxanthohumol, xanthohumol, and 8-prenylnaringenin, throughout the analytical process and to reduce the urine matrix effect in LC-ESI-MS/MS assays. It was concluded that a neutral pH (pH 7.0) is necessary for the analysis of prenylflavanoids, in order to maintain the stability of compounds for at least 24 h. The addition of ascorbic acid to the media improved stability, calibration curves, coefficients of correlation, accuracy, and precision parameters. Mix-mode cation exchange sorbent yielded the best matrix effect factors and recoveries. Method validation results showed appropriate intraday and interday accuracy and precision (<15%). Recovery of isoxanthohumol, xanthohumol, and 8-prenylnaringenin was 97.1% ± 0.03, 105.8% ± 0.05, and 105.4% ± 0.04, respectively, and matrix effect factors were nearly 100%. The stability assay showed that analytes were stable for at least 24 h. The method was applied to quantify 10 human samples of urine and was able to quantify prenylflavanoids in urine after the consumption of a single dose of beer (330 mL).