The purpose of this work was to develop a TLC-densitometric method for simultaneous identification and quantitative determination of azithromycin, clarithromycin, roxithromycin, spiramycin, and troleandomycin. The method was developed on TLC aluminium plates precoated with silica gel F 254 using solvent system izopropanol: n-hexane:ammonia 25% (8:12:3, v/ v/ v), which gives compact spots for azithromycin ( R F = 0.65), clarithromycin ( R F = 0.54), roxithromycin ( R F = 0.49), spiramycin ( R F = 0.22), and troleandomycin ( R F = 0.36). Densitometric analysis was carried out at 478 nm after spraying with (1:4, v/ v) sulphuric acid:ethanol and heating at 100°C for 5 min. The linear regression analysis data for the calibration plots showed good linear relationship with correlation coefficient higher than 0.99. The method is distinguished by high sensitivity, with limit of detection (LOD) from 0.34 μg/spot for troleandomycin to 0.67 μg/spot for clarithromycin and limit of quantification (LOQ) from 1.02 μg/spot for troleandomycin to 2.04 μg/spot for clarithromycin and a wide linearity range from 2 to 12 μg/spot for spiramycin and 2–15 μg/spot for other antibiotics. The precision of the determination was good; relative standard deviation (RSD) varied in the range from 1.49% to 4.14%.