<p>This dissertation focused on the development of mass spectrometric methodologies, separation techniques, and engineered devices for the optimal analysis of complex mixtures relevant to the energy sector, such as alternative fuels, petroleum-based fuels, crude oils, and processed base oils. Mass spectrometry (MS) has been widely recognized as a powerful tool for the analysis of complex mixtures. In complex energy samples, such as petroleum-based fuels, alternative fuels, and oils, high-resolution MS alone may not be sufficient to elucidate chemical composition information. Separation before MS analysis is often necessary for such highly complex energy samples. For volatile samples, in-line two-dimensional gas chromatography (GC×GC) can be used to separate complex mixtures prior to ionization. This technique allows for a more accurate determination of the compounds in a mixture, by simplifying the mixture into its components prior to ionization, separation based on mass-to-charge ratio (<i>m/z</i>), and detection. A GC×GC coupled to a high-resolution time-of-flight MS was utilized in this research to determine the chemical composition of alternative aviation fuels, a petroleum-based aviation fuel, and alternative aviation fuel candidates and blending components as well as processed base oils.</p> Additionally, as the cutting edge of science and technology evolve, methods and equipment must be updated and adapted for new samples or new sector demands. One such case, explored in this dissertation, was the validation of an updated standardized method, ASTM D2425 2019. This updated standardized method was investigated for a new instrument and new sample type for a quadrupole MS to analyze a renewable aviation fuel. Lastly, the development and evaluation of a miniaturized coreflood device for analyzing candidate chemically enhanced oil recovery (cEOR) formulations of brine, surfactant(s), and polymer(s) was conducted. The miniaturized device was used in the evaluation of two different cEOR formulations to determine if the components of the recovered oil changed.