Laying chickens were fed medicated feed containing various concentrations of sodium salinomycin (SAL) for 14 days followed by a 3 day withdrawal period. Eggs, collected during treatment and withdrawal, tissues and ovarian yolk of birds slaughtered after 0, 1, and 3 days’ withdrawal were extracted and analysed by high performance liquid chromatography (HPLC). Tissues, ovarian yolk and freeze‐dried egg albumen and yolk were extracted with acetone, followed by partitioning with petroleum ether and HPLC analysis. Albumen was extracted with methanol and analysed without further clean‐up. Salinomycin was detected at 520 nm after post‐column reaction with vanillin at 95°C. Recoveries of fortified salinomycin from freeze‐dried eggs (albumen and yolk) and tissue, premix and feed were nearly quantitative (>90%), except liver which was <85%. The detection limit was estimated to be 5 ng g−1, with the practical quantifiable limit being about 10 ng g−1. Highest SAL concentrations were in the more fatty components such as egg yolk, ovarian yolk and subcutaneous fat. SAL residues in other tissues were generally low and followed the order liver, kidney, thigh and breast muscles. SAL residues were dependent on the SAL concentration in feed and declined rapidly during withdrawal.