This study aimed to validate an accurate, precise and simple method. followed green chemistry to extract lincomycin from its liposomal coat and determined its levels in the serum and tissues of broiler chickens. Liposomes are natural compounds used for drug delivery. Nanomaterials with antibiotics aim to improve antibiotic effects and reduce side effects. The global trend follows the green analytical chemistry guidelines for the development of analytical methods to quantify materials. The extraction depended on ultracentrifugation of serum samples and solvent extraction with low centrifugation power for tissue extraction. A C18 column with an isocratic mobile phase consisting of acetone to acidified HPLC water with glacial acetic acid (2%) in the following ratio (16:84) was used. UV detector was set at 210 nm to detect lincomycin. The method had a short retention time of 3.227 min. The relative standard deviation (RSD) was < 2%. High recoveries ranged from 90.2 up to 102.1% in the different extracted biological samples. The low limits of detection were 0.025 and 0.2 µg/gm, which measure the high sensitivity level of the validated method and quantification ranged from 0.077 to 0.67 µg/gm. The pooled RSD for robustness of the lincomycin assay did not exceed 3.3 %. This method follows green chemistry, which is in agreement with economic requirements of developing countries. It was efficient for the separation of lincomycin from the nanoliposomecoat. It is selective and sensitive, reliable, reproducible, precise, and accurate according to the guidelines for the validation of the analytical methods.