It is currently unclear how the process of fat digestion occurs in the mouth of humans. This pilot study therefore aimed to quantify the levels of lipolytic activity at different sites of the mouth and in whole saliva. Samples of whole saliva and from 4 discrete sites in the oral cavity were collected from 42 healthy adult participants. All samples were analyzed for lipolytic activity using two different substrates (olive oil and the synthetic 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6’-methylresorufin) ester (DGGR)). Bland–Altman analyses suggested that the two assays gave divergent results, with 91% and 23% of site-specific and 40% and 26% of whole-saliva samples testing positive for lipolytic activity, respectively. Non-parametric multiple comparisons tests highlighted that median (IQR) of lipolytic activity (tested using the olive oil assay) of the samples from the parotid 20.7 (11.7–31.0) and sublingual 18.4 (10.6–47.2) sites were significantly higher than that of whole saliva 0.0 (0.0–35.7). In conclusion, lipolysis appears to occur in the oral cavity of a proportion of individuals. These findings give a preliminary indication that lipolytic agent activity in the oral cavity may be substrate-specific but do not discount that the enzyme is from sources other than oral secretions (e.g., microbes, gastric reflux).