BACKGROUND: The isolation and purification of vitamins A, E, D and cholesterol from food and feed test materials, for quantitation, is currently a time consuming and labor-intensive process. It includes separate steps for saponification, extraction, purification, and solvent evaporation. A new instrument (FLEX) was developed which improves and automates all steps involved, and which uses solid phase extraction (SPE). This study validates the FLEX automated method. OBJECTIVES: The objective of this study was to validate the automated method by recovery of standards, analysis of reference materials, comparison against proficiency test materials, and comparison against manual reference methods. METHODS: The FLEX instrument automatically adds reagents, mixes and heats to saponify test materials, filters the digestate, extracts with SPE, and evaporates solvent. RESULTS: The accuracy of the automated FLEX Instrument method was confirmed by the agreement with NIST reference materials for retinol, α-tocopherol, cholecalciferol, ergocalciferol, and cholesterol. Accuracy also was compared against manual reference methods on 11 different food types that ranged from 4-100% fat, 0-75% protein, and 0-85% carbohydrates. The automated and manual methods were highly correlated with no bias or distortion over the range of test materials. Precision was similar to the manual methods for retinol recovery but improved for α-tocopherol and cholecalciferol analysis. The accuracy of the automated method also was confirmed for feed analysis. Eleven different animal feeds were analyzed in the FLEX and results were highly correlated with Association of American Feed Control Officials proficiency test results. CONCLUSIONS: The automated method accurately and efficiently performed the multiple analytical steps necessary for the isolation and purification of the analytes in preparation for chromatographic analysis. HIGHLIGHTS: The automated method was compared against industry standard methods and yielded equivalent results and improved precision. SPE technology was optimized to efficiently elute non-polar analytes, while retaining protein and other medium-polar analytes.