Petroprophyrins are biomarkers used to extract information about petroleum genesis among other characteristics. Identification of particular types, such as Ni, Cu, Mn, vanadyl (VO), and oxygenated or sulfur-containing porphyrins, typically involves exhaustive isolation and purification processes followed by high-resolution mass spectrometry analysis using atmospheric pressure photoionization [APPI-(+)] or electrospray [ESI-(+)] sources. Simultaneous identification of all porphyrins present in a particular crude oil or organic-matter-rich sediment still remains an analytical challenge. Here, we report a straightforward petroporphyrin isolation and identification methodology based on a single-step liquid–liquid (L–L) extraction (crude oil: acetonitrile) and high-performance thin-layer chromatography fractionation (HPTLC, aminopropyl-bonded silica) followed by selective ionization via electron transfer in matrix-assisted laser desorption ionization (MALDI-FTICR). Mass spectrometric analysis of the extracts resulted in detection of 350 individual compounds in the acetonitrile extract and 518 in the HPTLC extract, corresponding to the porphyrin families N4VO, N4VO2, N4VO3, N4VOS, and N4Ni as verified by isotopic structure analysis. To the best of our knowledge, this observation constitutes the largest simultaneous identification of Ni, VO, and oxygenated and sulfur-containing porphyrins in a single crude oil sample. In addition, the use of MALDI significantly reduces the amount of sample required for analysis (pico to femtomole levels) in comparison with continuous infusion methods such as APPI and ESI.