An anxiolytic fraction of Tilia americana standardized in tiliroside, rutin, quercitrin, quercetin glucoside, and kaempferol was obtained. After oral administration of the fraction, the above-mentioned flavonoids were not detected in plasma over 24 h. However, meta and para hydroxyphenylacetic acid and dihydroxyphenylacetic acid (m-HPAA, p-HPAA and DOPAC) were monitored. These are the biotransformation compounds of the aglycones of kaempferol and quercetin; these aglycones are products of the other flavonoids present in the anxiolytic fraction. The analytical methods (HPLC) for flavonoids and the related compounds (m-HPAA, p-HPAA and DOPAC) were validated, determining the parameters of accuracy, precision, specificity or selectivity, limit of detection, quantification range, and robustness. The pharmacokinetic assay was performed with ICR mice strains, which were given 200 mg/kg of the standardized active fraction. The results of validation of the analytical methods were obtained, allowing it to be established in a validated way that no flavonoids present in the anxiolytic fraction of T. americana were detected in plasma. However, detection and follow up was possible for the serum levels of m-HPAA, p-HPAA, and DOPAC. The three compounds follow a two-compartment model with very similar parameters between m-HPAA and p-HPAA, some being different from the ones characterized in the pharmacokinetics of DOPAC.