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Due to the potential speed and flexibility of surface plasmon resonance (SPR)-biosensor analysis, the technique is currently being exploited for detection of residues in food. In the present study an antibody-based inhibition assay for analysis of clenbuterol in cattle hair was developed using a Biacore 2000 instrument. Analysis of clenbuterol with and without enhancement with a secondary antibody was compared. A fast extraction method for hair, which enabled direct analysis in the biosensor with no clean up other than microscale ultrafiltration was developed. In contrast to most of the published methods in the area, no organic solvent or solid phase extraction step was needed. The biosensor method was validated by analysis of hair samples from animals treated with clenbuterol and the results were compared to results from GC-MS analysis. The correlation was good (r2=92.7%). The detection limit of the new method was 10 ng g−1 hair.