Bioanalytical strategies for the quantification and characterization of novel bioconjugate therapeutics have been thoroughly discussed over the past several years .Typical approaches for ADC quantification in support of pharmacokinetic assessments entail monitoring surrogate analytes (peptides/payloads) via a targeted bottom-up approach. Therefore, information on the biotransformation can be lost without a priori knowledge. High resolution accurate mass spectrometry (HRMS) based intact analysis of ADCs coupled with chromatographic separation is a powerful and robust tool for the identification of novel biotransformation species. Recent advances in the field of HRMS in addition to more efficient ionization of macromolecules enable the progress of analyzing intact biotherapeutics such as mAbs and ADCs . However, other approaches such as CE, HIC and SEC coupled with MS have been employed as well. Han et al. reported case studies with CE-MS applied to protein biotransformation analysis. He et al. pioneered in ADC biotransformation with RPLC-MS approaches. Additional applications of HIC-MS and SEC-MS suggest that alternative approaches can be explored for structural analyses of ADCs. AZD8205 is a B7-H4-targeted ADC utilizing a novel topoisomerase I linker-payload (Figure ) being studied in the clinic for the treatment of biliary tract, breast, ovarian or endometrial cancers (NCT05123482) . As part of the structure-activity relationship (SAR) optimization of AZD8205 we examined 4 different linkers to enable the conjugation of the topoisomerase I payload (TOP1i AZ14170132) . The payload was covalently conjugated to native interchain cysteines of an anti-B7-H4 antibody via either a caproyl or propionyl-PEG8 spacer to a Val-Ala (VA) or Gly-Gly-Phe-Gly (GGFG) peptide linker (Figure ), resulting in four distinct anti-B7-H4 ADCs, each with an approximate drug-toantibody ratio (DAR) of 8. To characterize AZD8205 pharmacokinetics and biotransformation using both in vitro incubation and in vivo plasma samples in mice dosed with AZD8205, we employed intact and bottom-up approaches. Herein, we describe comprehensive characterization of pharmacokinetics and biotransformation of an ADC from both in vitro and in vivo samples, employing orthogonal approaches providing complementary information. The findings confirmed durable structural and conjugation stability of AZD8205 among the 4 linker designs evaluated.