High-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) are powerful tools for the characterization of the molecular structures of components of both simple and complex mixtures. MS and MS/MS have played key roles in many fields, including proteomics, metabolomics, and petroleomics. This thesis focuses on the development of tandem mass spectrometric methods for the structural characterization of asphaltenes and small isomeric molecules. In addition, this thesis also presents a method to address a sampling bias in asphaltene analysis. Chapter 2 describes the fundamental aspects of the mass spectrometer used for the research discussed in this thesis. Chapter 3 presents an in-depth study on the determination of the relative abundances of single-core and multicore compounds in asphaltenes. A statistical sampling bias in many earlier asphaltene studies is discussed and a rapid and simple alternative method is introduced to address this sampling bias. Chapter 4 presents an investigation of the sizes of aromatic cores and lengths of alkyl chains in isobaric ions derived from asphaltenes by using an improved data analysis method. Chapter 5 presents a fundamental study on utilization of energy-resolved collision-activated dissociation (CAD) for the differentiation of isomeric ions. Energy-resolved CAD was used to compare the ability of two different activation methods to differentiate four sets of isomeric ions of aromatic hydrocarbons. The comparison suggests that medium-energy CAD (MCAD; commercially known as higher-energy collision dissociation, HCD) is more powerful in differentiation of the four sets of isomers than conventional ion trap CAD (ITCAD)