In this paper, reliability and validity of a recently in-house developed dereplication strategy based on LC-MS and LC-UV/Vis PDA techniques were verified. The dereplication strategy was applied to investigate the alkaloid rich fraction of Colchicum brachyphyllum Boiss. & Haussk. ex Boiss. (Colchicaceae) which has been previously analyzed phytochemically. Both studies results, LC-MS and LC-PDA and phytochemical analysis, were matched in seven compounds namely: (-)-colchicine (5), 3-demethyl-(-)-colchicine (4), (-)-cornigerine (7), beta-lumi-(-)-colchicine (6), (-)-demecolcine (3), 3-demethyl-(-)-demecolcine (2), and 2,3-didemethyl-(-)-demecolcine (1). LC-MS and LC-PDA based dereplication strategy was not able to detect two of the compounds that were reported in the phytochemical study namely: (+)-demecolcinone and (-)-androbiphenyline. This finding could raise a question about the "naturality" of (+)-demecolcinone. On the other hand apigenin (8) was identified only using this dereplication strategy. Four compounds which had molecular ions at m/z 372 and 400 in the stems, and at m/z at 372 and 358 in flowers, could not be dereplicated, and were thus considered unknown. Their spectral data were not matched with neither the compounds that have been isolated previously from this species nor with any other colchicinoid; hence they should be pursued as potential new compounds.