Rats regularly undergo surgical procedures that may result in pain. Alleviation of unnecessary pain is an ethical and regulatory responsibility. Buprenorphine is an opioid analgesic commonly used in rats and requires dosing every 6 to 8 h to be effective. Frequent administration is time consuming and may increase stress, post-surgical pain, and dehiscence in rats, making the use of long-acting formulations an attractive alternative. A transdermal buprenorphine solution (TBS), FDA approved for use in felines, is commercially available and effective for up to 96 h. We hypothesize that a single dose of TBS in rats will result in clinically relevant plasma buprenorphine concentrations (greater than 1 ng/mL) for up to 96 h. To test this, 39 rats were randomly assigned to the following treatment groups: low dose (LD; 5 mg/kg; n = 6 females, 6 males), high dose (HD; 10 mg/kg; n = 6 females, 6 males), and vehicle control (CON; n = 7 females, 8 males). TBS or anhydrous ethanol (CON) were topically applied. Blood was collected at 4, 24, 72, 96, and 168 h postadministration, and buprenorphine concentrations were determined via HPLC-MS. To quantitatively assess adverse effects, daily fecal output, food intake, and body weight were measured, and observations of hematuria and skin lesions were documented. Plasma buprenorphine concentrations exceeded 1 ng/mL in all TBS rats at 4, 24, 48, and 72 h. No rats experienced serious adverse effects or developed gross lesions at the application site. The HD group had decreased fecal output compared with CON. Both TBS groups had reduced weight gain compared with CON. These results suggest that TBS dosed at 5 to 10 mg/kg could provide analgesia for up to 3 d in rats, and administering a lower dose mitigates some adverse effects.