The aim of this in-vivo study was to compare total protein and four key salivary proteins present in the acquired enamel pellicle (AEP) on eroded and non-eroded surfaces in participants with erosive tooth wear. Participants with erosive tooth wear of dietary non-intrinsic origin, present on the occlusal surfaces of the lower first molars and an unaffected posterior occlusal surface in the same quadrant were recruited from restorative dental clinics at King's College London Dental Institute (n = 29, REC ref 14/EM/1171). Following removal of the salivary film, AEP samples were collected from the eroded occlusal surfaces (EP, n = 29) and the non-eroded occlusal surfaces (NP, n = 29) using 0.5% sodium dodecyl sulfate (SDS) soaked filter papers. Total protein concentration was analysed using bicinchoninic acid assay (BCA). Protein fractions were separated using SDS-PAGE and immunoblotted against: mucin5b, albumin, carbonic anhydrase VI (CA VI) and statherin antibodies. Amounts were quantified using ImageLab software against purified protein standards of known concentration. ANOVA followed by paired t-test and Wilcoxon's matched-pair signed-rank test were used to test statistical significance. The difference was considered to be significant at a P value < 0.05. The total protein on eroded surfaces was significantly lower compared to the total protein on non-eroded surfaces [0.41mg/mL (0.04) and 0.61 mg/mL (0.11)] respectively (p< 0.05). The median (min, max) amount of statherin was also significantly lower on eroded occlusal surfaces [84.1 (20.0, 221.8) ng] compared to AEP from non-eroded teeth in the same subjects [97.1(30.0, 755.6) ng] (p = 0.002). No statistical differences were observed for mucin 5b, albumin or CA VI. The total protein and statherin in the in-vivo AEP were different between eroded and non-eroded tooth surfaces of the same patient.