Glycopeptide antibiotics are widely used in the treatment of infections caused by Gram-positive bacteria. They inhibit the biosynthesis of the bacterial cell wall through binding to the D-alanyl-D-alanine (D-Ala-D-Ala) terminal peptide of the peptidoglycan precursor. Taking advantage of this highly specific interaction, we developed a direct fluorescence polarization based method for the detection of glycopeptide antibiotics. Briefly, we labeled the acetylated tripeptide Ac-L-Lys-D-Ala-D-Ala-OH with a fluorophore to create a peptide probe. Using three glycopeptide antibiotics, vancomycin, teicoplanin, and telavancin, as model compounds, we demonstrated that the fluorescence polarization of the peptide probe increased upon binding to antibiotics in a concentration dependent manner. The dissociation constants (K(d)) between the peptide probes and the antibiotics were consistent with those reported between free d-Ala-d-Ala and the antibiotics in the literature. The assay is highly reproducible and selective toward glycopeptide antibiotics. Its detection limit and work concentration range are 0.5 microM and 0.5-4 microM for vancomycin, 0.25 microM and 0.25-2 microM for teicoplanin, and 1 microM and 1-8 microM for telavancin. Furthermore, we compared our assay in parallel with a commercial fluorescence polarization immunoassay (FPIA) kit in detecting teicoplanin spiked in human blood samples. The accuracy and precision of the two methods are comparable. We expect our assay to be useful in both research and clinical laboratories.