AbstractReputable calculations such as the Friedewald formula are used extensively to determine LDL‐cholesterol (LDL‐C) values from known total cholesterol, triacylglycerol and HDL‐cholesterol (HDL‐C) levels. To the best of our knowledge, however, the validity of this equation has not yet been confirmed in rats. The aim of the present study is to give some insights as to why this formula must be used carefully in rats, and to find cut‐off points below which this formula can be considered reliable. Sera of 54 rats with different cholesterol, triacylglycerol and HDL‐C levels were tested. LDL was isolated by ultracentrifugation and LDL‐C measured by an enzymatic colorimetric method and compared against LDL‐C obtained by the formula. In rats whose serum cholesterol was <100 mg/dL, or whose HDL‐C constituted ≥75% of total cholesterol, or whose cholesterol/phospholipids ratio was <1, or whose serum did not contain β‐VLDL, LDL‐C obtained by both methods did not significantly differ. Under other conditions, however, and particularly in hypercholesterolaemic rats who did present β‐VLDL, the results clearly show that the Friedewald formula overestimates LDL‐C levels. In conclusion, (VLDL + LDL)‐C instead of VLDL‐C and LDL‐C must be used when ultracentrifugation or other alternative methods are not available to measure LDL‐C in hypercholesterolaemic rats.