To characterize the levels of 11 inflammatory mediators (IFNg, IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17, TNF, and VEGF) in saliva samples from NB on the first and second day of hospitalization in the Neonatal Intensive Care Unit (NICU). Exploratory study, descriptive, nested within a primary clinical, observational, and prospective study, conducted in the NICU of a public hospital in São Paulo, Brazil. Demographic data and vital signs were recorded in the clinical records of 90 NB, and five saliva samples from 5 NB were collected between the first and second day of life (D1-D2) at approximately 8-hr intervals (8-9 am, 4-5 pm, and 11-12 pm). Saliva samples were used for the measurement of 11 cytokines (IFNg, IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17, TNF, and VEGF). Five NBs participated in this exploratory study, and the vital signs showed variability from the first (D1) to the second day (D2) of hospitalization, variability similar to that of the total population of the primary study. The presence and levels of the 11 cytokines were detected in the saliva samples, as well as a statistical correlation between 10 cytokines (IFNg, IL1b, IL2, IL4, IL6, IL10, IL12, IL17, TNF, and VEGF) and vital signs. The novelty of measuring inflammatory mediators in saliva samples from hospitalized NBs in the NICU is highlighted, providing support and new perspectives for the development of clinical and experimental research and an opportunity for developing and implementing new salivary biomarkers in different population segments.