A thin layer chromatographic-densitometric method for identification and quantitation of neomycin sulfate, polymixin B sulfate, zinc bacytracin and auxiliary substances (methyl and propyl hydroxybenzoates) in ophthalmic ointment was developed. To separate these constituents the silica gel coated TLC plates and two mobile phases were used. The suitable mobile phases were: methanol-n-butanol-ammonia 25%-chloroform (14:4:9:12, v/v/v/v) for determination of antibiotics and n-pentane-glacial acetic acid (66:9, v/v) for methyl and propyl hydroxybenzoates. The antibiotic chromatograms were detected by using ninhydrin ethanol solution, while densitometric measurements were made at lambda = 550 nm. Hydroxybenzoates were identified by UV measurements at lambda = 260 nm. The constituents under consideration were well separated at sufficient detection level. The recovery for all constituents ranged from 98.08% to 104.95%.