For over a decade there has been an intensive debate on the possible natural origin of boldenone (androst-1,4-diene-17β-ol-3-one, 17β-boldenone) in calf urine and several alternative markers to discriminate between endogenously formed boldenone and exogenously administered boldenone have been suggested. The currently approved method for proving illegal administration of β-boldenone(ester) is the detection of β-boldenone conjugates. In the presented method the sulphate, glucuronide and free fractions are separated from each other during cleanup on a SAX column to be able to determine the conjugated status of the boldenone metabolites. The sulphate and glucuronide fractions are submitted to hydrolysis and all three fractions are further cleaned up on a combination of C18/NH2 solid-phase extraction (SPE) columns. Chromatographic separation of the boldenone metabolites was achieved with a Waters Acquity UPLC™ instrument using a Sapphire C18 (1.7 µm; 2 × 50 mm) column within 5 min. Detection of the analytes was achieved by electrospray ionisation tandem mass spectrometry. The decision limits of this method, validated according to Commission Decision 2002/657/EC, were 0.08 ng mL−1 for androsta-1,4-diene-3,17-dione, 0.13 ng mL−1 for androst-4-ene-3,17-dione, 0.11 ng mL−1 for 17α-boldenone, 0.07 ng mL−1 for 17β-boldenone, 0.24 ng mL−1 for 5β-androst-1-en-17β-ol-3-one and 0.58 ng mL−1 for 6β-hydroxy-17β-boldenone. Because of the fractionation approach used in this method there is no need for conjugated reference standards which often are not available. The disadvantage of needing three analytical runs to determine the conjugated status of each of the metabolites was overcome by using fast chromatography. Copyright © 2008 John Wiley & Sons, Ltd.