Characterization of challenging and/or new energy materials (e.g., biomass/biofuels, heavy oils, and solid petroleum products) requires the generation of new and improved analytical technologies. Matrix-assisted laser desorption/ionization (MALDI) and laser desorption/ionization (LDI) have enabled the characterization of various petroleum fractions and products that were previously unanalyzable via mass spectrometry (MS). Bypassing the solvent restrictions normally imposed when using traditional ionization approaches (e.g., atmospheric pressure photoionization), laser-based ionization techniques allow for the direct analysis of solid and non-volatile petroleum products. Understanding the molecular complexity of heavy oil and its refinery products is required for more effective processing and utilization of crude oil, the optimization of downstream processes, and the development of new petroleum-based products. This review highlights the analytical challenges that hinder the characterization of crude oil and its refined products and how both MALDI and LDI MS have been utilized to characterize and better understand the molecular compositions of these samples. We will focus in particular on the sample preparation and characterization of heavy resid including the vacuum distillation resid, asphaltenes, petroporphyrins, petroleum pitch, and briefly, new algae-based biofuel research.