We established a convenient assay method for measuring elongation of very long chain fatty acids (ELOVLs) using a Unifilter-96 GF/C plate. The Unifilter GF/C plate preferentially interacts with hydrophobic end products of ELOVLs (i.e., long chain fatty acid), with minimal malonyl-CoA (C2 unit donor for fatty acid elongation) interaction. This new method results in the quick separation and detection of [(14)C] incorporated end products (e.g., [(14)C] palmitoyl-CoA) from reaction mixtures containing excessive amounts of [(14)C] malonyl-CoA. In the Unifilter-96 GF/C plate assay, recombinantly expressed human ELOVLs (i.e., ELOVL1,-2,-3,-5 and -6) displayed appreciable assay windows (>2-fold vs. mock-transfected control), enabling us to conduct comprehensive substrate profiling of ELOVLs. The substrate concentration profile of ELOVL6 in the Unifilter-96 GF/C plate assay is consistent with that obtained from the conventional liquid extraction method, thus, supporting the reliability of the Unifilter-96 GF/C plate assay. We then examined the substrate specificities of ELOVLs in a comprehensive fashion. As previously reported, ELOVL1, -3 and -6 preferably elongated the saturated fatty acyl-CoAs while ELOVL2 and ELOVL5 preferentially elongated the polyunsaturated fatty acyl-CoAs. This further confirms the Unifilter-96 GF/C plate assay reliability. Taken together, our newly developed assay provides a convenient and comprehensive assay platform for ELOVLs, allowing investigators to conduct high density screening and characterization of ELOVLs chemical tools.