The presence of tetracycline (TC) antibiotics was determined in animal feed that had been previously screened with a microbiological plate test. Feed samples were screened by a microbiological plate test on a pH 6.0 culture medium seeded with Bacillus cereus ATCC 11778 able to pre-reveal the presence of tetracyclines. Subsequently, confirmation and quantification were performed using a validated HPLC method with mass spectrometric detection. In 2013–2018, 353 feed samples were analysed to detect antibacterial substances, of which 186 (52.7%) were suspected to contain tetracyclines. Forty-two out of 186 (22.6%) samples analysed by the chromatographic method contained undeclared tetracyclines, which were determined at concentrations from 0.3 to 49 mg kg−1. The most frequently identified contaminating tetracyclines were doxycycline and chlortetracycline.