The analysis of molecular cues in dentinal fluid from an excavated cavity could improve diagnostics in the context of minimally invasive caries treatment. In the current clinical trial we assessed whether the dentinal fluid levels of MMP-9 (matrix metalloproteinase-9; neutrophil gelatinase) would increase with the progression of carious lesions. MMP-9 is associated with neutrophil-related tissue breakdown in the pulp. Absolute MMP-9 levels were contrasted against the levels of MMP-2, an enzyme related to normal tissue turnover. Dentinal fluid was collected below deep and shallow caries from molars and premolars within the same patients aged 18 years and older (n = 30, 1 tooth per group/patient). Experimental teeth were isolated under a rubber dam prior to excavation. Dentinal fluid was collected from the bottom of the cavity using a size 25 paper point. MMP levels were assessed using an enzyme-linked immunosorbent assay. Nonparametric methods were applied to test for differences between groups. Significantly more (p < 0.05, Wilcoxon test) MMP-9 was collected from the deep carious lesions than from the shallow counterparts. Pairwise comparison of MMP-9 values within patients revealed that there was more MMP-9 collected from deep lesions than from shallow counterparts in 27 of the 30 individuals under investigation (pairwise Wilcoxon test, p < 0.001). In contrast, no such difference existed for MMP-2. There was a high correlation between MMP-9 from deep and shallow lesions (Spearman's ρ = 0.72, p < 0.001), indicating that patients with more MMP-9 in the deep carious lesion also tended to have more MMP-9 in the shallow lesion.