In the present study our earlier studied bioactive ether insoluble phenolic components from n-butanol fraction (EPC-BF) of defatted flaxseed meal were chemically characterized by HPTLC and LC-ESI-MS. HPTLC profile and fingerprinting were recorded by using silica gel plates and then separation was achieved by using solvent system butanol: water (5:4, v/v). Chromatogram was developed at 310 nm after spectral scanning from wavelength 190 to 700 nm. The HPTLC plate was derivatized with Folin- Ciocalteu reagent. Similarly, for LC-ESI-MS analysis, separation of EPC-BF was carried out on C18 column with use of isocratic mobile phase methanol: water (65:35, v/v) at set detector wavelength 310 nm. An electrospray ionization mass spectrum was recorded at negative mode over m/z 100-1500. The results of HPTLC and LC-ESI-MS characterization of EPC-BF tentatively confirmed the presence of phenolic acids such as caffeic acid, prototannin, hydrolysable tannin in EPC-BF of defatted flaxseed meal along with phenolic glycosides.