Abstract: The separation, identification, and determination of epimer of tetracycline have been attempted paper-chromatographically based on the principle of Selzer's method. Some improvements were made to effect an efficient separation and identification. It was demonstrated experimentally that results from Selzer's method were not reliable owing to partial inversion of tetracycline in the course of chromatography. However, better results could be obtained through the modification of the solvent system. In order to determine the epimer quantitatively, the chromatogram concerned was cut down and eluted many times with 0.1 N sulphuric acid. The optical density of the elute was then determined spectrophotometrically at 355 mμ, and finally by using E_1cm^1%=333 the quantity of epimer was worked out accordingly.