Bitumen is a complex mixture corresponding to the residue of the vacuum distillation of crude oil. It can be separated on the basis of polarity and solubility with saturates, aromatics, resins, and asphaltenes (SARA) fractionation. High-performance thin-layer chromatography is suited to analyze complex mixtures such as petroleum products, which separates compounds according to their polarity. In this work, the direct coupling between HPTLC and laser desorption ionization (LDI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR) was developed and optimized for the characterization of bitumen. Silica gel and cellulose stationary phases were tested to separate bitumen samples. A higher signal-to-noise ratio (S/N) was obtained with cellulose, whereas an irreversible adsorption phenomenon was observed with silica gel. With cellulose as a stationary phase, the bitumen sample was separated into two zones using a mixture of heptane and ethanol 80:20 (v/v). In addition, manual fractionation of bitumen with heptane was realized to use the fractions as standards. The separation of bitumen was underlined by the difference of ion distribution between the two zones and the formation of fullerene ions on the noneluted zone. Indeed, N-2, N1S2, N2S1, and N2O1 classes were mainly found in the noneluted zone, whereas HC, O-1, O-2, and O1S1 classes were mostly found on the eluted zone. Additionally, the DBE versus C# maps highlighted that the compounds with the higher DBE values were on the noneluted zone. These results as well as the manual fractionation of bitumen sample allowed one to confirm that the noneluted zone corresponded to asphaltenes and the eluted zone corresponded to maltenes. Overall, this study showed a fast separation and characterization of asphaltene and maltene fractions using HPTLC separation.