While few methodological studies have been published on the salivary measurement of malondialdehyde (MDA), none have precisely detailed the analytical method. This work presents the development of an analytical method for MDA measurement in microvolumes of saliva samples from healthy individuals, using thiobarbituric acid derivatization and fluorescence reading of the formed compound. This method was progressively designed to meet specific constraints such as the limited sample volume available, cost-effectiveness of each assay, time required for analysis and costs. After studying the impact of various parameters, quantitative validation of this method was conducted using the accuracy profile. The study demonstrated that the fluorometric analysis method for salivary MDA accurately quantifies the concentration of this molecule within a validity range from 0.031 μM to 2.3 μM. Additionally, the accuracy profile allowed estimation of measurement uncertainty for each concentration level. It was determined that the calibration method using raw data was most appropriate as it introduced the least uncertainty in the obtained results.