UNLABELLED: BACKGROUND: Owing to its ease of collection, saliva is potentially the sample of choice in diagnosis. Salivary biomolecules have provided a porthole in surveying a person's health and well-being. Our study aims were (1) to demonstrate the effects of pre-analytical steps, collection and pre-processing techniques on salivary protein detection and (2) to establish an indication of salivary reference intervals for 3 biomolecules of clinical interest. METHODS: Saliva samples were collected from participants (n = 25, ages 20-35 years) using the following methods: no stimulation (resting/unstimulated), mechanical, and acid stimulation. The saliva was prepared for analysis by: unprocessed, post standard centrifugation in a container without any additives, and centrifugation using Centrifugal Filter Unit (Amicon® Ultra-0.5). AlphaLisa® assays were used to measure the levels of C-Reactive Protein (CRP), Immunoglobin (IgE) and myoglobin in saliva samples. RESULTS: Saliva flow rates were lowest with the resting/drooling collection method. The lowest total protein concentration was with acid stimulation. Unstimulated and mechanically stimulated collections produced no effect on the CRP and IgE levels while myoglobin levels were highest with the unstimulated collection. Acid stimulation had a negative impact on the measured concentrations of IgE and myoglobin (except for CRP levels). CONCLUSION: Mechanical stimulation was the most viable option for collecting saliva without affecting the levels of CRP and myoglobin. The processing methods had an adverse effect on the concentration of total protein as well as on CRP and IgE concentrations.