Abstract Ramoplanin is a novel glycolipodepsipeptide antibiotic, currently undergoing clinical trials. This method describes the determination of ramoplanin by direct injection of human urine into a coupled-column liquid chromatographic system. An internal-surface reversed-phase column has been used for on-line sample clean-up and enrichment. Analytical separation of ramoplanin and MDL 62 456 used as internal standard, has been achieved on a ABZ+ reversed-phase column with ammonium acetate buffer–acetonitrile–methanol according to a gradient profile. Analytes were detected by their UV absorbance at 270 nm. The limit of quantitation was 0.1 μg/ml urine and the limit of detection was found to be 0.035 μg/ml, corresponding to 13.7 pmol/ml. Linearity was determined in the range 0.1–2 μg/ml. Precision (relative standard deviation) ranged from 0.71 to 8.75% and the accuracy from −9.9 to 11.6%. Different human sources were tested and no interference between analytes and urine constituents was observed. The method is simple and rapid, requiring a total analysis time of 35 min per sample and reaching greater selectivity and accuracy than microbiological assays.