In the current proposed analysis, a new, feasible, and selective fluorimetric approach was designed for baclofen assay. Baclofen is a medication prescribed as a therapy for muscle spasticity that originated from multiple sclerosis or a spinal cord injury and other cases as hiccups. The analytical approach relies on the utility of ninhydrin for formation of fluorescent derivative that was monitored at (λ 386 nm, λ 480 nm). At suitable reaction conditions, the primary amino moiety in baclofen is condensed with ninhydrin and phenylacetaldehyde in the presence of Teorel buffer as a buffered medium. The method exhibited linearity when baclofen concentration plotted against response in the range (1 – 10 μg mL ). Adjustment of the reaction variables and studying validation parameters according to directives of ICH were performed perfectly. Moreover, an interference study was implemented to ensure that no discrepancy from the excipient. Finally, the proposed method was applied successfully for baclofen assay in dosage form and extended to test Mylobac content uniformity.This article is protected by copyright. All rights reserved.