Recent FDA and ICH guidances on safety testing of drug metabolites have challenged the way we traditionally think about quantitative bioanalytical methods. Such assays, in general, require a reference standard for each analyte to construct calibration curves and prepare quality control samples. However, early in the drug development process, metabolite standards may not be readily available, and if they are inherently unstable, they are difficult to synthesize or purify. In this paper, we describe a novel in-line method for producing and then quantifying a very unstable metabolite which is based upon the in situ postcolumn coulometric oxidation of the parent drug. Lacking any metabolite standards, the feasibility of simultaneously quantifying a development drug (compound A) and its unstable hydroxylated metabolites (metabolite B) was investigated. Reference standards for these ostensibly major human metabolites could not be reliably obtained due to rapid degradation upon purification and/or subsequent storage. Following high-performance liquid chromatography (HPLC) separation, parent drug and its [(13)C(3)-(15)N] isotopically labeled internal standard were quantitatively converted to equal amounts of a diastereomeric pair of hydroxylated metabolites using a postcolumn coulometric electrochemical cell before reaching the mass spectrometer. The concentration of the injected parent (which is equal to the total concentration of the in-line generated metabolites since the conversion to metabolite is quantitative) and the tandem mass spectrometry (MS/MS) signals of the electrochemically generated metabolites were used to construct a calibration curve for quantifying both the parent drug and its hydroxylated metabolites. Plasma extracts from humans dosed with compound A contained chromatographically distinct liquid chromatography-mass spectrometry (LC-MS) signals (m/z 538) for in vivo formed hydroxylated metabolites and the electrochemically oxidized parent drug which had been converted in-line into its chemically identical twin. Both peaks in this study sample could be quantified using a single calibration curve obtained under the same coulometric conditions using known amounts of the parent drug. Although no attempt was made to fully validate a bioanalytical method, the practicality of this in situ quantification approach was further confirmed by the preliminary bioanalytical analysis of a selection of plasma samples collected following oral administration (50 mg) of compound A in a clinical study.