This study introduces the know-how of preparing a multiple injection form atropine sulphate solution. An injectable aqueous solution of atropine sulphate at a concentration of 0.1%. was prepared under aseptic conditions in dark glass bottles each containing 50 ml. The preparation was intended for animal use only. It contained 1g atropine sulphate, 9 g sodium chloride as a normal saline, benzyl alcohol 15 ml as a preservative and water for injection up to 1000 ml. The pH of the solution was adjusted to 4.2 (range 3.0-6.5). The preparation of 0.1% atropine sulphate solution was clear colorless solution free from undesired particles. It complied with the requirements for injectable solutions. Further, the preparation was safe when used under laboratory conditions in chicks, rats and donkeys. It was also effective in preventing dichlorvos (an organophosphate insecticide)-induced poisoning in chicks in a manner comparable to a commercial preparation of 0.1% atropine sulphate. In conclusion, the know-how of a preparation of 0.1% atropine sulphate solution is presented for veterinary use. [Vet. World 2012; 5(3.000): 145-149