In the pharmaceutical industry, LC-MS has been generally applied for quantitative (bio)analysis, biomarker analysis, impurity profiling, and metabolite identification. This dissertation focuses on the development, validation, and application of LC-MS methods in the bioanalysis of small and large molecules and is largely divided into two categories: bioanalysis of thiamine, its metabolites and analogs, and metabolite identification of therapeutic oligonucleotides. Chapter 1 of the thesis comprises the introduction section and gives a general overview of LC-MS methods in association with the studies investigated in the dissertation. The chapter therefore contains the prefaces of two categories, which helps to orient readers on the purpose of the research described in the dissertation. Chapter 2 presents a validated LC-UV method for the bioanalysis of two lipophilic thiamine analogs, benfotiamine and sulbutiamine, in cancer cells. Chapter 3 focuses on the development and validation of an LC-MS method for the simultaneous determination of stable isotope-labeled and unlabeled thiamine, thiamine monophosphate, and thiamine pyrophosphate in cancer cells. Chapter 4 describes the metabolite identification of an investigational therapeutic oligonucleotide, eluforsen, using an LC-MS method. Finally, Chapter 5 presents an LC-MS method for the determination of the impact of chemical modifications of oligonucleotides on their metabolic stability.