Trachyspermum ammi Linn. Sprague (Fam. Umbelliferae), commonly known as Bishop’s weed (English names) and ajwain in Indian languages. It is an erect annual herb with striate stem. The seeds are hot, pungent, stomachic, appetizer, aphrodisiac, carminative, laxative and diuretic. In present studies, the author extracts essential oil by hydro-distillation for 4‑5h in Clevenger apparatus. The present study reports physicochemical characterization and spasmolytic activity of essential oils from ajwain (Trachyspermum ammi Linn.) in Rajasthan. The ajwain fruits were hydro-distilled for 4‑5h in Clevenger apparatus. Physicochemical characterization of the extracted oils was carried out to determine their solubility, yield%, acid value (mg/KOH/g), saponification value (mg/KOH/g), ester value, iodine value (g/g), and peroxide value (mEq/kg). The gas chromatographic and Thin Layer chromatographic analysis were also conducted. Data regarding the yield and physicochemical properties of yellowish, brown oil have been reported. Antispasmodic activity was assessed on isolated guinea pig ileum. Effects of acetylcholine, fruits extract and acetylcholine along with fruits extract were studied on isolated guinea pig ileum; which later compared with standard anti-spasmodic agent. The present study results revealed that essential oil of Trachyspermum ammi Linn. Sprague (Fam. Umbelliferae) showed promising antispasmodic action on excised guinea pig ileum.