The effects of filtration (F) and alpha-amylase depletion (AD) were assessed in n = 34 saliva samples. Each saliva sample was split into three aliquots and treated as follows: (1) no treatment; (2) 0.45μm commercial filter; and (3) 0.45μm commercial filter and affinity depletion of alpha-amylase. Then, a panel of biochemical biomarkers consisting of amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), creatine kinase (CK), calcium, phosphorus, total protein, albumin, urea, creatinine, cholesterol, triglycerides, and uric acid was measured. Differences between the different aliquots were observed in all measured analytes. The most marked changes were found in triglycerides and lipase data for filtered samples, and in alpha-amylase, uric acid, triglycerides, creatinine, and calcium results in alpha-amylase-depleted aliquots. In conclusion, the salivary filtration and amylase depletion methods employed in this report caused significant changes in saliva composition measurements. Based on these results, it would be recommended to consider the possible effects of these treatments in salivary biomarkers when filtration or amylase depletion is performed.