A novel, simple, fast, and miniaturized method, termed in-line micro-matrix solid-phase dispersion (in-line MMSPD), coupled with high performance liquid chromatography (HPLC) was developed for the simultaneous extraction and determination of Sudan dyes (i.e. Sudan I-IV, Sudan orange G, Sudan black B, and Sudan red G) with the aid of an experimental design strategy. In this method, a matrix solid-phase dispersion (MSPD) column including a suitable mixture of polar sorbents was inserted in the mobile phase pathway, and while the interfering compounds were retained, the analytes were eluted and entered into the analytical column. In this way, the extraction, elution, and separation of the analytes were performed sequentially. Under the optimal experimental conditions (including the amount of sample, 0.0426 g; amount of dispersant phase, 0.0216 g of florisil, 0.0227 g of silica, 0.0141 g of alumina; and blending time, 112 s), the limits of detection (LODs), limits of quantification, linear dynamic ranges, and recoveries were obtained to be 0.3-15.3 mu g kg(-1), 1-50 mu g kg(-1), 50-28,000 mu g kg(-1), and 94.5-99.1%, respectively. The results obtained showed that determination of the selected Sudan dyes in food samples using an enough sensitive and a simple analytically validated method like in-line MMSPD may offer a suitable screening method, which could be useful for food analysis and adulteration. (C) 2015 Elsevier B.V. All rights reserved.