A sensitive and rapid HPLC-assay for determining the new oxazolidinone antibiotic linezolid in serum and urine is described. HPLC-integrated sample preparation permits the direct injection of serum and urine samples without any pre-treatment. The in-line extraction technique is realized by switching automatically from the extraction column to the analytical column. After the matrix has passed the extraction column the retained analyte will be quantitatively transferred to the analytical column where separation by isocratic HPLC will be performed. Linezolid is detected according to its absorption maximum at 260 nm. The quantification limits are estimated to be 0.3 and 0.5 μg/ml in serum and urine samples, respectively. The described procedure allows sample clean-up and determination of the antibiotic within 20 min, thereby facilitating drug-monitoring in clinical routine.