Ketamine (KT) is a chiral anesthetic agent, ( R )- and ( S )-enantiomers of which differ in their pharmacological properties. KT has become one of the most commonly used illicit drugs in the world, thus, rapid and feasible on-site testing is required to crack down on the illicit use. Although immunochemical approach with specific antibodies is promising for this purpose, in practice anti-KT antibodies are difficult to obtain. We here disclose generation of monoclonal antibodies against KT. Mice were immunized with either (a) commercially-available or (b) in-house-prepared KT-albumin conjugates. Splenocytes from these mouse groups (a and b) were separately fused with P3/NS1/1-Ag4-1 myeloma cells. After standard screening and cloning, we established 5 hybridoma clones: 2 were derived from group-a mice [generating Ab-KT(a)#2 and #37] and 3 were from group-b mice [generating Ab-KT(b)#9, #13, and #45]. These antibodies exhibited practical performance in competitive enzyme-linked immunosorbent assay systems. When (±)-KT·hydrochloride (HCl) was used as the competitor, dose–response curves showed midpoint values of 30 and 70 ng/assay (a-series antibodies) and 2.0–3.0 ng/assay (b-series antibodies). Remarkably, the a-series antibodies were specific for ( S )-KT·HCl, while the b-series antibodies were specific for ( R )-KT·HCl. Ab-KT(a)#2 ( K _a, 7.5×10⁷ M⁻¹) and Ab-KT(b)#45 ( K _a, 7.7×10⁸ M⁻¹) exhibited the highest enantioselectivity for each group, and cross-reactivity with the ( R )- and ( S )-antipodes was 1.3 and 1.7%, respectively. The hybridomas established here are also valuable as a source of genetic information for the anti-KT antibodies, which is required for progressing to next-generation technologies using genetically engineered antibodies.