A rapid, sensitive and simple optimized spectrophotometric method is developed for the determination of Glucosamine (GL) based on reaction of GL with sodium 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline medium forming orange colored product which measured at 451 nm. The method optimization was essential as many procedural parameters influenced the efficiency and reproducibility of the derivatization process including the reaction time, reaction temperature,NQS concentration, alkali concentration and solvents. The objective of our study was to optimize and validate a spectrophotometric method using the reaction of GL with NQS for determining GL in dosage form. The factors that may influence the efficiency and reproducibility of the derivatization process were identified and optimized. The proposed method was validated and successfully applied for the analysis of GL in dosage form. Statistical comparison of the results of the proposed method with the reference method showed excellent agreement and proved that no significant difference in the accuracy and precision. The developed method is easy to use, accurate and least cost-effective for routine studies relative to HPLC and other techniques