Gastrodin (GAS) and its aglycone, p-hydroxybenzyl alcohol (HBA), are both bioactive compounds extracted from Gastrodia elata Blume (GEB). In the current Chinese pharmacopoeia, they are regarded as quality control markers for GEB. In this study, we developed a high-performance liquid chromatography method coupled with a diode array detector to quantify GAS and HBA concentrations in plasma following oral ingestion by rats. For the first time, GAS was detected in vivo after HBA administration. GAS and HBA both had similar pharmacological effects, but the influence of the glucose moiety resulted in different pharmacokinetic characteristics. In this study, the effects of GAS and HBA at different administration durations were investigated in zebrafish larvae. These compounds were found to induce a sedative effect but had different onset times. In conclusion, a biotransformation of HBA to GAS could be observed in the rats. This may be a new insight into the pharmacokinetic characteristics of these bioactive compounds and also relates to the different ways in which they take effect.