This in vitro study investigated the possible dehydration of dentin caused by bleaching agents. Furthermore, it tested whether protective dentin varnishes can maintain the physiological moisture of dentin during bleaching treatment. Fifty-five standardized dentin cylinders were prepared from freshly extracted bovine incisors under constant water irrigation. Prior to bleaching, the treatment specimens were conditioned at room temperature in a hygrophor for 14 days. The samples were divided into 11 groups. The Group A specimens, which were completely dehydrated, and Group B, which was stored for 2 weeks in a hygrophor, served as controls (A, B n=5). The other samples (n=10 each group) were coated with Vivasens [VS] (C), Bilfuorid [BF] (D) and Seal&Protect [SP] (E). Five specimens from each group (C-E) were subsequently treated with an experimental bleaching gel (Exp BG) (20% carbamide peroxide [CP], glycerine-based gel): Cb, Db, Eb. The remaining specimens were bleached with Exp BG (F) only, Vivastyle (G: 16% CP, glycerine-based gel) or Vivastyle Paint On (H: 6% CP-varnish) for 7 days (n=5 each group) with bleaching time for gels: 2 hours/day, paint on: 20 minutes/day. After the respective treatments, the overall water content of each specimen was determined using the analytical method of Karl-Fischer-titration. The water content of bovine dentin (Group B, mean%+/-SD) obtained in this study amounted to 15.24+/-0.4. All bleaching products significantly reduced the water content compared to the controls (exp BG: 13.32+/-0.47, Vivastyle 13.2+/-0.27, paint on 13.72+/-0.54; p<0.05). Also, application of SP before bleaching resulted in reduced water content (14.06+/-0.12; p=0.0005). However, bleaching with exp BG following use of VS (14.99+/-0.42) or SP (13.85+/-0.26) did not result in a reduction of water content in dentin. Pretreatment with BF did not protect dentin from water loss during bleaching (12.44+/-0.38; bi p=0.0009). All glycerine-based bleaching products used in this study had a significant dehydrating effect on dentin. The application of protective varnishes prior to bleaching treatment may reduce or even prevent dentin dehydration.