Densitometric high performance thin layer chromatography (HPTLC) quantification method was developed to validate the decolorization/biotransformation of Disperse Orange ERL and dye mixture by lichen Parmelia sp. which release several colored compounds during decolorization process, hence unable to use colorimetric estimation. Percent decolorization of Disperse Orange ERL and dye mixture by lichen Parmelia sp. was observed when estimated using developed HPTLC method. Limit of detection and limit of quantification for both dyes in mixture were obtained as 03 and 1 mu g/mu l, respectively. Area of peak of control Disperse Orange ERL was reduced by 43% after 12 h, 71% after 48 h and upto 82% after 72 h of incubation. Precision and repeatability of data elucidated the % relative standard deviation less than 3 for all the values thus indicating statistically acceptable. Biodegradation of dye and mixture was confirmed with Fourier transform infrared spectroscopy analysis, i.e., altered fingerprinting spectral pattern. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.