A microbiological method for determining traces of antibiotics in low volume biological samples, which comprises determining the minimum inhibitory concentration (MIC) of an antibiotic selected from a group consisting of minocycline, ciprofloxacin, kanamycin, tetracycline and oxacillin against Staphylococcus aureus. The bacteria are grown overnight in Mueller-Hinton broth (MHB), said cultures subsequently being diluted to 0.5 McFarland standard (1.5 x 108 cells/mL). Said antibiotics are then added to 96-well plates to a final volume of 200 µL and incubated at 37°C for 18 hours. Absorbency is determined and the results are expressed as the percentage of inhibition relative to the control containing the biological sample, selected in turn from urine, blood, synovial fluid, eye aqueous humour or cerebrospinal fluid (CSF), and the bacterial culture. The curve representing the percentage of bacterial growth inhibition versus concentration of antibiotic is obtained, and fitted by "Four Parameter Logistic Curve (4PL)" analysis, yielding the antibiotic concentration values in the biological sample. The method allows to determine amounts of antibiotic in the biological sample even at ng/mL level.