Background and Aims: Heterozygous Familial Hypercholesterolemia (HeFH) is a common genetic disease responsible for premature atherosclerosis. Therefore, early diagnosis and treatment are recommended to reduce cardiovascular risk. The screening is usually based on plasma LDL-cholesterol. In children, blood testing is often an obstacle for screening. This study aims to evaluate the performance and feasibility of a salivary non-invasive screening of HeFH in a pediatric population. Methods: The salivary cholesterol of 30 HeFH pediatric patients and 30 healthy age-matched controls was measured using 2 different enzymatic kits: Kit-1 (Amplite™ Kit - AAT Bioquest®) and Kit-2 (Total Cholesterol Assay Kit - CELL BIOLABS®, Inc) Results: The median serum LDL-cholesterol in controls and HeFH patients was 2.3 and 4.1 mmol/L respectively (p<0.001). The median serum total-cholesterol in control and HeFH patients was 3.8 and 5.7 mmol/L respectively (p<0.001). Using successively Kit-1 and Kit-2, the median salivary total-cholesterol was respectively 0.5 μmol/L and 2.4 μmol/L in controls and 0.7 μmol/L and 3.0 μmol/L in HeFH patients (p=0.3). However, no correlation was found between salivary and serum cholesterol concentrations neither in the total population (rho index = - 0.03, p=0.8, for Kit-1 and rho index = 0.1, p= 0.3, for Kit-2) nor in controls and in HeFH patients. Conclusions: Although cholesterol is detectable in saliva, no correlation was found between saliva and plasma cholesterol with the 2 kits. As easy as saliva collection appears, the processing seems more demanding than serum.