Rhamnus alaternus L. (Rhamnaceae) is a medicinal plant traditionally used for treating a range of inflammatory diseases. This study aimed to characterize the methanolic extract of Rhamnus alaternus L. stem bark (MRAB) using LC-MS and to investigate its in vitro antioxidant activity as well as its in vivo analgesic, anti-inflammatory, and wound-healing effects. Thirteen compounds were identified, including 7 glycosylated flavonoids, two non-glycosylated flavonoids, two phenolic acids and two chromones. The IC50 of MRAB for DPPH and ABTS radical scavenging activity were 58.31 ± 3.07 μg/mL and 22.33 ± 0.35 μg/mL, respectively. MRAB also demonstrated a high total antioxidant capacity and reducing power (242.8 ± 17.96 mg Asc E/g extract and 143.74 ± 1.18 mg Asc E/g extract, respectively). The MRAB showed notable analgesic and anti-inflammatory effects. In the acetic acid-induced abdominal writhing test, MRAB at 500 mg/kg exhibited a significant reduction in writhing (66.19 %), comparable to diclofenac (76.66 %). The extract also showed potent analgesic effects in the formalin test, with a 79.54 % inhibition in the inflammatory phase, similar to salicylic acid (84.9 %). MRAB exhibited strong anti-inflammatory activity in both the carrageenan-induced hind paw edema test (58.74 % and 66.73 % inhibition at 250 and 500 mg/kg, respectively) and the xylene-induced ear edema test (49.72 % and 59.81 % inhibition). Furthermore, MRAB indicated significant wound-healing effects, with retraction percentages of 89.56 ± 0.93 % and 93.45 ± 0.83 % at 250 and 500 mg/kg, respectively, comparable to the Cicatryl group (95.91 ± 0.62 %) on day 12. These findings highlight the therapeutic potential applications of MRAB for analgesic, anti-inflammatory, and wound-healing treatments.