A method that integrated thin-layer chromatography (TLC) with femtosecond laser ablation-inductively coupled plasma-mass spectrometry (fs-LA-ICP-MS) was developed for the determination of the proportionality of vanadium/nickel in the asphaltene fraction of crude oils. TLC is easy and fast to implement for separation of crude oil components requiring minimal amount of solvents, and fs-LA-ICP-MS allows for direct analysis of the TLC plates without any additional sample preparation. This method was tested on Venezuelan crude oil samples and their isolated asphaltenes. The results correlated well to those obtained using a traditional method for asphaltene analysis involving separation and digestion with measurements by inductively coupled plasma-optical emission spectroscopy (ICP-OES). The combination of TLC with fs-LA-ICP-MS provided fast and reliable measurements of V/Ni proportionality in asphaltene and directly from the crude oil without solvent-exchange processes.