Cathepsin K is a cysteine proteinase, primarily expressed in osteoclasts, which has a strong collagenolytic activity and plays an essential role involved in bone matrix degradation. Its inhibition could provide a novel approach to the treatment and prevention of osteoporosis. One structural class of lead compounds in our cathepsin K inhibitors program is based on an arylaminoethyl amide scaffold, which has potential metabolic weak points that might be stabilized by appropriate chemical modification(s). For the identification of potential metabolic "soft spots" and the rational design of improved derivatives, early biotransformation of a potent arylaminoethyl amide cathepsin K inhibitor (NVP-AAV490-NX) was investigated in plasma, urine and liver homogenates of rats after intravenous bolus administration of 10 mg/kg. The detection and identification of metabolites was achieved by high-resolution mass spectrometry (time-of-flight MS) and multi-dimensional mass spectrometry (ion trap MS). Both mass spectrometers were combined with reversed-phase capillary high-performance liquid chromatography columns. It was demonstrated that both mass analyzers complement each other and that, even in the sub-nanogram range, the resulting set of MS data can be successfully used to elucidate most of the metabolic changes unambiguously, solely by mass spectrometric techniques. The proposed metabolite structures were additionally corroborated by exact mass measurement of the protonated molecular ions to confirm the predicted elemental composition, by determination of the number of the exchangeable hydrogen atoms replacing water against deuterium oxide as mobile phase and, in one case, by an MS(3) product ion experiment in order to elucidate the site of conjugation.