Yoghurt is a popular dairy product in Iran because of its beneficial influence on human health and nutritional value. Aflatoxin M1 (AFM1) is the metabolite of potential carcinogen aflatoxin B1, which can contaminate milk through the feed and is not eliminated by common processing heat treatment. An analytical method using immunoaffinity column for extraction and a high-performance liquid chromatography (HPLC) for quantification was developed for AFM1 in this study. An HPLC method with fluorimetric detection for the determination of AFM1 in yoghurt milk has been optimized and validated according to Commission Decision BS EN ISO 14501: 2007 by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CCα) and detection capability (CCβ) of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401: 2006:EC. A new HPLC method with fluorescence detection was developed to determine aflatoxin M1. The detection limit was 1 ng/kg for yoghurt. The calibration curve was linear from 0.1 to 3.0 μg l⁻¹ injected. The method includes a preliminary clean-up and the average recoveries determined on three different days at the concentration levels of 0.025, 0.050 and 0.075 μg kg⁻¹ were in the range of 72.57%-86.66% with RSD in the range of 2.56%-8.41%. The interday and interlevel mean recovery value, which has been used to correct routine analysis results, was 80%. The method is rapid, easily automatable and therefore useful for accurate and precise screening of aflatoxin M1 in yoghurt.