In addition to having hallucinogenic properties desired by its users, GHB has been used to render sexual assault victims semiconscious, physically vulnerable, and amnestic. It is categorized as a Schedule I substance under the Controlled Substance Act. GBL is a corresponding cyclic ester of GHB and can easily hydrolyze back to GHB in aqueous solutions. GBL is classified only as a List I chemical; however, if ingested, it could be treated as a Schedule I substance under Federal law. Gas chromatography analyses are the most popular means for measuring GHB in forensic samples. Although sensitive, these methods require the chemical conversion of GHB into the volatile GBL or silyl derivatives. Other techniques include Fourier Transform infrared spectroscopy, ion mobility spectrometry, and high-performance liquid chromatography. Detection with these methods still suffers from requiring additional steps such as extraction and the use of buffered solutions. Thus, they are not suitable for investigating the GBL-GHB equilibrium. Nuclear magnetic resonance spectroscopy (NMR) is a recognized tool for the direct detection of a wide range of small endogenous or exogenous molecules in complex biofluids such as urine and serum; therefore, NMR is ideal for direct monitoring of the GBL-GHB equilibrium. This paper reports on the effective use of 1H NMR for quantifying the relative amount of GHB and GBL in various buffered solutions. It also describes spike experiments that involved human urine and serum samples, so as to determine the detection limit for GHB. The materials and methods used are described. The testing showed no esterification of GHB at any pH. 7 figures and 12 references