RBD1016 is an -acetylgalactosamine-conjugated siRNA drug currently in a phase II trial for treatment of chronic hepatitis B virus. To evaluate its absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic/pharmacodynamic (PK/PD) properties, two LC-based bioanalytical methods, LC-high-resolution/accuracy MS and LC-fluorescence detection, were developed and qualified. The LC-high-resolution/accuracy MS method was used for metabolite identification and simultaneous quantitation of the antisense and sense strands as well as their respective metabolites. The LC-fluorescence detection assay was primarily used for analyzing the antisense strand and its metabolites in low-concentration plasma samples. The two methods were successfully bridged by analyzing the same sets of study samples. Both methods were found to have excellent accuracy/precision, specificity and reproducibility to support ADME and PK/PD studies of RBD1016 siRNA.