A new sample preparation method, stir bar sorptive extraction (SBSE), has been evaluated for the enrichment of organic solutes from biological fluids such as urine and blood. In SBSE, a stir bar coated with a polydimethylsiloxane layer is stirred for a given time in the sample. After sampling the stir bar is placed in a thermal desorption unit coupled on-line to capillary gas chromatography-mass spectrometry (SBSE-TD-CGC-MS). The principle and operation of SBSE are presented. Total profiling and target compound analysis have been selected as applications to illustrate the performance of SBSE-TD-CGC-MS (MSD). It is demonstrated that a variety analytes ranging from biological markers (phenols, hormones, fatty acids) to artificial contaminants (recreational drugs, plasticizers) can be enriched with high sensitivity. For polar solutes, in-situ derivatization can enhance both recovery into the polydimethylsiloxane (PDMS) layer and chromatographic analysis. Two types of derivatization have been applied, derivatization with ethyl chloroformate and with acetic acid anhydride. Linearity, detectability, and repeatability are illustrated by the determination of 1-hydroxypyrene in a urine sample from a smoker.