Aflatoxins are toxic metabolites produced by Aspergillus spp. Because aflatoxins are potent carcinogens in humans and animals, many countries have set regulatory limits for aflatoxins in foods to prevent dietary exposure. From a global food safety perspective, in 2023, the Codex Alimentarius Commission established the maximum level (ML) of total aflatoxins in certain cereals and cereal-based products, including polished rice. Therefore, validated analytical methods for aflatoxin detection are necessary. In this study, a high performance liquid chromatography (HPLC)-fluorescence method coupled with multifunctional column clean-up and trifluoroacetic acid derivatization was developed for the determination of aflatoxin levels in polished rice. Our method was validated in a single laboratory study using aflatoxin-spiked materials, followed by an inter-laboratory validation study. Twelve laboratories participated in the inter-laboratory validation study, and five polished rice test samples artificially contaminated with aflatoxins were analyzed. In a single laboratory study, the ranges of mean recoveries of aflatoxin B1, B2, G1, G2, and total aflatoxins were 101%, 100-103%, 93-96%, 95-98%, and 97-99%, respectively. The relative standard deviations for within-day and between-day variations were all ≤ 4.4%. In the inter-laboratory validation study, the relative standard deviations for repeatability and reproducibility were from 0.7 to 2.7% and 3.3 to 8.9% for all analytes, respectively. In response to the Codex ML and method performance criteria for aflatoxins in polished rice, an analytical method based on HPLC-fluorescence detection was developed. All method performance parameters estimated from the testing results of the single-laboratory and inter-laboratory validation studies met the criteria required by the Codex. Single- and inter-laboratory studies for the validation of an analytical method for aflatoxin level determination in polished rice were successfully performed. This analytical method will be suitable to determine aflatoxin levels around the Codex ML set for polished rice.