This study aimed to assess the effect of biomodification before adhesive procedures on the tooth-restoration interface of class V restorations located in caries-simulated vs. sound dentin, and the quality of dentin surface by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Class V cavities located on cervical dentin were prepared on the buccal surfaces of extracted human molars under the simulation of intratubular fluid flow. Two dentin types, i.e., sound and demineralized by formic-acid, were biomodified with 1% riboflavin and calcium phosphate (CaP) prior to the application of a universal adhesive (Clearfil Universal Bond) in etch and rinse or self-etch mode, and a conventional micro hybrid composite (Clearfil APX). Restorations were subjected to thermo mechanical fatigue test and percentages of continuous margins (% CM) before/after fatigue were compared. Bio modification of dentin surfaces at the molecular level was analyzed by Time-of-Flight Secondary Mass Spectometry (ToF-SIMS). % CM were still significantly higher in tooth-restoration interfaces on sound dentin. Meanwhile, biomodification with riboflavin and CaP had no detrimental effect on adhesion and in carious dentin, it improved the % CM both before and after loading. Etching carious dentin with phosphoric acid provided with the lowest results, leading even to restoration loss. The presence of molecule fragments of riboflavin and CaP were detected by ToF-SIMS, evidencing dentin biomodification. The adhesive interface involving carious dentin could be improved by the use of a collagen crosslinker and CaP prior to adhesive procedures.