Objectives: This study was designed for preliminary phytochemical screening of Citrullus lanatus var. citroides fruit pulp secondary metabolites. In addition, the polyphenolic compounds of the methanolic extract (ME) and its n-butanol soluble fraction (n-BF) were analyzed, and the in vivo analgesic activity of the richest polyphenolic fraction was evaluated. Methods: TheMEwas phytochemically screened for various secondary metabolites. Moreover, ME and n-BF polyphenolic profiling was performed using high performance liquid chromatography (HPLC) against external standards. The analgesic activity of the polyphenolic rich fraction (n-BF) was evaluated in vivo using acetic acid-induced abdominal writhing and hot plate tests. Results: Preliminary phytochemical screening revealed the presence of carbohydrate and /or glycosides, flavonoids, saponins, tannins, and unsaturated sterols and /or triterpenes, while alkaloids and/or compounds containing nitrogenous bases, anthraquinones, cardiac glycosides, and coumarins were absent. HPLC polyphenolic profiling showed the tentative identification of 11 phenolic compounds in ME (19.6959%), including 6 phenolic acids and 5 flavonoids, with gallic acid and rutin being the major identified ones; respectively. As well as, 17 phenolic compounds were tentatively identified in the n-BF (75.0771%), including 10 phenolic acids with chlorogenic acid as the major identified one, and 7 flavonoids with naringenin as the major one.Then-BF showed significant peripheral and central analgesic activity in a dose-dependent manner (p < 0.05), with n-BF at a dose of 1000 mg/kg being the most potent. Conclusion: The present study revealed that the n-BF was highly rich in phenolic compounds. These phenolic compounds were reported for the first time in C. lanatus var. citroides. The n-BF showed significant analgesic activity which was evaluated for the first time for the plant.