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An accurate, sensitive, precise, reliable, and quick method for the determination of cholesterol content by high-performance thin layer chromatography is developed. In this method, aluminum-backed precoated silica gel 60 F 254 plates were used as the stationary phase and the samples were sprayed with the help of CAMAG sample applicator Linomat 5. The chromatogram was developed with the mobile phase consisting of chloroform:methanol (9.5:0.5, v/v). The samples were detected using CAMAG Scanner 4 and evaluated using the method developed on winCATS software. Densitometric analysis of cholesterol was performed in absorbance mode at 200 nm. In this solvent system, cholesterol gave a compact spot with an R f value of 0.63 ± 0.03. The linear regression analysis of data for the calibration curve showed good linearity over a concentration range of 2-7 μg/spot with a regression value of 0.99933 and standard deviation of 1.44%. The limit of detection and limit of quantification were found to be 100 ng/spot and 500 ng/spot, respectively. Using the developed method, the concentration of cholesterol in the saponified and unsaponified egg yolk sample was determined. This method was found to be reproducible and can even be used for samples containing complex matrices., (Copyright © 2014. Published by Elsevier B.V.)