Phytosterols reduce cholesterol absorption and low-density lipoprotein (LDL) cholesterol concentrations, but the quantity and physiological significance of phytosterols in common diets are generally unknown because nutrient databases do not contain comprehensive phytosterol data. The primary aim of this study was to design prototype phytosterol-deficient and high-phytosterol diets for use in controlled feeding studies of the influence of phytosterols on health. A second aim was to quantify the phytosterol content of these prototype diets and three other diets consumed in the United States. This study was conducted from June, 2001 to September, 2008 and involved designing, preparing, and then analyzing five different diets: an experimental phytosterol-deficient ‘control’ diet, a relatively high-phytosterol diet based on the Dietary Approaches to Stop Hypertension (DASH) diet, American Heart Association (AHA) diet, Atkins® lifetime maintenance plan, and a vegan diet. A single day of meals for each diet was homogenized and the resulting composites were analyzed for free, esterified, and glycosylated phytosterols by gas chromatography. Independent samples t tests were used to compare the diets’ total phytosterol content. The total phytosterol content of the experimental phytosterol-deficient diet was 64 mg/2000 kcal, with progressively larger quantities in Atkins®, AHA, vegan, and the high-phytosterol DASH diet (163, 340, 445 and 500 mg/2000 kcal, respectively). Glycosylated phytosterols, which are often excluded from phytosterol analyses, comprised 15.9 ± 5.9% (mean±SD) of total phytosterols. In summary, phytosterol-deficient and high-phytosterol diets that conform to recommended macronutrient guidelines and are palatable can now be used in controlled feeding studies.