The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices.