The complete spectrum of active compounds of Hypericum perforatum (St. Johns wort) has not yet been fully elucidated, but some naphthodianthrones (hypericin and pseudohypericin), phloroglucinols (hyperforin) and flavonoids (primarily quercetin glycosides) are thought to be essential for the antidepressant activity of its extracts. Some of these compounds modulate the expression of the P-glycoprotein transporter or of cytochrome P450 enzymes or both although, again, their real role in clinically relevant interactions involving the extracts and conventional drugs is still not clear. Analysis of main components in body fluids and tissues has thus become of interest in studies to identify and characterize those accounting for the primary and secondary pharmacological effects, which can help in providing safe and effective dosing regimens with the appropriate extracts. Selective procedures such as high-performance liquid chromatography with fluorimetric, electrochemical, ultraviolet or photodiode detection or coupled with mass spectrometry, depending on the constituent, have been frequently used. This review provides an overview of these methods, and the related sample pretreatment procedures. The focus is also on their sensitivity and precision and consequently their reliability for pharmacokinetic studies in relation to the blood and, when animal data are available, brain concentrations of the main active components of Hypericum perforatum extracts.