Fluorescence spectroscopy with the spectral resolution routine PARAFAC is a leading tool to analyze Natural Organic Matter (NOM) in waters. This routine resolves spectra into humic-, fulvic- and protein-like components, which helps interpret the NOM dynamics in environmental systems. This work is one of the first systematic studies dealing with the impact of chemical perturbations on the fluorescence spectral interpretation of NOM. The samples, taken at two Canadian Shield locations (a shallow set and a deep set to ∼650 m), were perturbed for pH (‘titrations’ from pH 4 to 10) and salinity (from ∼0.02 to 3‰ salt content), then analyzed by fluorescence/PARAFAC. Our fluorescence signals for the three components showed no clear change with pH, as would be expected with a classic titration. The signals were reproducible between replicates for the humic- and protein-like components, but less so for the fulvic-like components. Changes of salinity only had a small impact on the fluorescence signal (a ∼2.7–3.4% signal decrease for each salinity unit, ‰) for the three components in this salinity range.