A Quality by Design approach was used to develop a sensitive spectrophotometric method for determination of glucosamine Sulphate in pharmaceutical dosage forms and optimize a derivatization reaction of glucosamine with o-phthalaldhyde. This strategy was focused on derivatization process understanding and analytical method development and validation. Both of them were evaluated by applying risk assessment and Design Space tools. The scouting step was carried out by analyzing different derivatizing agents, type of buffers, pH of buffers, and time of reaction. Five critical process parameters (CPPs) were selected related to, pH of borate buffer, volume of 2-mercaptoethanol (ME), volume of o-phthalaldhyde (OPA), and the time of reaction. The effect of different levels of CPPs on critical quality attributes (CQAs) (the absorbance of derivative) was evaluated in the screening design using fractional factorial design. Using response surface methodology and contour plots the optimum values of the selected factors were determined and the design space of the method was defined. Finally, control strategy was designed and the method was validated and applied to a real sample of glucosamine dosage forms using the optimum derivatization conditions of the reaction; pH of borate buffer 10.5, concentration of buffer 0.05M, Time of reaction 10.7 minutes, volume of ME 100μL, and volume of OPA 5.1mL at room temperature. The results of the present study clearly shown that the developed method was sensitive relative to the other published spectrophotometric and HPLC methods with identification of the most significant factors affecting the derivatization reaction.