This study aims to compare the single-dose pharmacokinetic profiles of semaglutide administered via sublingual, oral, and injectable routes in Sprague-Dawley rats. Semaglutide was delivered sublingually in a proprietary anhydrous suspension vehicle. Rats were randomized into five groups and received the following treatments: subcutaneous injection (0.011 mg/kg), sublingual suspension (1 mg/kg, prepared from either commercial tablets or peptide powder), and oral tablets (1 mg/kg and 20 mg/kg). Semaglutide was detectable in plasma within 2 minutes post-dosing in all groups except the oral 1 mg/kg group. Sublingual administration demonstrated lower variability in plasma concentrations compared to oral dosing. At 1 mg/kg, the sublingual route achieved a significantly higher area under the curve (AUC) than oral (82.53 vs.15.08 ng*h/ml, p=0.004), indicating improved bioavailability. The maximum plasma concentration (C) was reached within 30 minutes for oral and sublingual routes, and at 8 hours for subcutaneous injection. The relative bioavailability was 0.06% for oral 1 mg/kg, 0.16% for oral 20 mg/kg, and 0.34% and 0.29% for sublingual 1 mg/kg using tablets or powder, respectively. No significant difference in AUC was observed between sublingual semaglutide prepared from oral tablets versus powder. These results highlight the potential of sublingual delivery of semaglutide and suggest this route may improve absorption while reducing variability. This proof-of-concept study supports further development of sublingual semaglutide formulations and pharmacokinetics research in humans.