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Antimicrobial drugs have been widely used in dairy industry for more than five decades generally to prevent or treat mastitis. The detection of antibacterial residues in milk requires screening methods that are simple, quick and sensitive at antibiotic concentrations close to the maximum residue limit (MRL). A new competitive receptor test TwinsensorBT was validated and compared with Delvotest®SP-NT an agar diffusion microbiological test. Both tests were designed for screening antimicrobial substances in milk. The performance criteria described by the Commission Decision 2002/657/EC, ISO 13969:2003, ISO 18330:2003 and Guide for analytical validation of screening methods (AFSSA Fougères) were used for the validation study. Validation was made on spiked samples of milk with 12 different β-lactams (penicillin-G, ampicillin, amoxicillin, cloxacillin, nafcillin, cefapirin, cefalonium, cefazolin, cefoperazone, ceftiofur, cefalexin, cefquinome) and 4 tetracyclines (doxycycline, chlortetracycline, oxytetracycline, tetracycline). The TwinsensorBT test was found to be easy to use, with very short incubation period (6 minutes), robust and sensitive to all certified β-lactame and tetracycline antibiotics at or lower concentrations than EU maximum residue limits, except for nafcillin. The Delvotest®SP-NT on other hand has a longer incubation period (3 hours) and is less sensitive to oxytetracycline, but it can detect a wide range of other antimicrobial substances mostly at or below EU MRLs. [ABSTRACT FROM AUTHOR]