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Enzymatic assays for cholesterol determination can use an end point or a kinetic method. We evaluated and compared the performance of these methods. We constructed user-defined methods on 3 automated analyzers using Streptomyces cholesterol reagents to evaluate the analytic performance of both methods. Linearity (700–900 mg/dL) and stability of reagents (5–11 weeks) depended on the analyzers. The coefficients of variation for imprecision were 2.41% to 2.99% and 3.78% to 5.52% for the end point and kinetic methods, respectively. The end point method showed less bias at decision cut points (−0.8% to 1.3%) than did the kinetic method (−1.1% to 3.6%) but was more affected by interfering substances.The advantages of the end point over the kinetic method are better precision and lower reagent cost. The end point imprecision fell within National Cholesterol Education Program guidelines (≤3%), but the kinetic did not. The kinetic method showed less interference and required shorter analysis time.