In vitro studies in transfected cells have indicated that chemical chaperones including glycerol (0.5–1.2 M) and trimethylamine oxide (TMAO, 50–100 mM) can correct defective trafficking of some proteins, including ΔF508 CFTR in cystic fibrosis and AQP2 mutants in nephrogenic diabetes insipidus. To develop a mouse model to test the efficacy of chemical chaperones in vivo, glycerol and TMAO were administered by intraperitoneal (i.p.), subcutaneous (s.c.), and oral routes. Glycerol and TMAO assays that utilized 1–5 μL of tail vein blood were developed. Administration by the i.p. and s.c. routes gave maximum serum glycerol concentrations of ∼100 mM, levels that were well below the effective in vitro concentrations. Single i.p. or s.c. doses of TMAO (7 g/kg, 8% solution in water) resulted in serum [TMAO] greater than 50 mM, with a long half-life (t 1/2 ∼18–21 h). Sustained high serum and tissue [TMAO] > 52 mM for 3 days was achieved by s.c. administration of TMAO (7 g/kg) in water every 8 h. Although ∼50% of the mice died with this multiple-dose regimen, the remaining mice had nearly normal liver, renal, and pancreatic function. A lower dose of TMAO (5 g/kg) given by the s.c. route every 8 h resulted in serum [TMAO] concentration of 22 mM, a level that was well tolerated by all mice for 72 h. These mice also had high [TMAO] in urine, 400 mM. These results demonstrate that potentially therapeutic concentrations of TMAO can be sustained in mice in vivo, permitting the testing of chemical chaperones in transgenic mouse models of diseases caused by defective protein trafficking.