In Burkina Faso, the values that serve as clinical chemistry reference ranges are those provided by European manufacturers’ insert sheets based on reference of the Western population. However, studies conducted so far in some African countries reported significant differences in normal laboratory ranges compared with those of the industrialized world. The aim of this study was to determine reference values of cholesterol fractions in apparently normal adults in Burkina Faso that could be used to better assess the risks related to cardiovascular diseases. Study population was 279 healthy subjects aged from 15 to 50 years including 139 men and 140 women recruited at the Regional Center of Blood Transfusion of Ouagadougou, capital city of Burkina Faso (West Africa). Exclusion criteria based on history and clinical examination were used for defining reference individuals. The dual-step precipitation of HDL cholesterol sub-fractions using dextran sulfate was performed according to the procedure described by Hirano. The medians were calculated and reference values were determined at 2.5th and 97.5th percentiles. The median and upper ranges for total cholesterol, LDL cholesterol, total HDL cholesterol and HDL2 cholesterol were observed to be higher in women in comparison to men (p <0.05). These reference ranges were similar to those derived from other African countries but lower than those recorded in France and in USA. This underscores the need for such comprehensible establishment of reference values for limited resources countries. Our study provides the first cholesterol sub-fractions (HDL2 and HDL3) reference ranges for interpretation of laboratory results for cardiovascular risk management in Burkina Faso.