Saliva is an interesting, non-conventional, valuable diagnostic fluid. It can be collected using standardized sampling device; thus, its sampling is easy and non-invasive, it contains a variety of organic metabolites that reflect blood composition. The aim of this study was to validate a user-friendly method for the simultaneous determination of low molecular weight metabolites in saliva. We have optimized and validated a high throughput, direct, low-cost reversed phase liquid chromatographic method with diode array detection method without any pre- or post-column derivatization. We indexed salivary biomolecules in 35 whole non-stimulated saliva samples collected in 8 individuals in different days, including organic acids and amino acids and other carbonyl compounds. Among these, 16 whole saliva samples were collected by a single individual over three weeks before, during and after treatment with antibiotic in order to investigate the dynamics of metabolites. The concentrations of the metabolites were compared with the literature data. The multianalyte method here proposed requires a minimal sample handling and it is cost-effectiveness as it makes possible to analyze a high number of samples with basic instrumentation. The identification and quantitation of salivary metabolites may allow the definition of potential biomarkers for non-invasive “personal monitoring” during drug treatments, work out, or life habits over time.