Objective: Doxycycline hyclate is a broad spectrum antibiotic with activity against a wide range of gram-positive and gram-negative bacteria and it is widely used as a pharmacological agent. A simple, selective, linear, precise and accurate ultraviolet detection (UV) method has been developed and applied for the determination of doxycycline hyclate in different pharmaceutical samples. Methods: Acid-base analysis and titrimetric method were utilized to determine the value of pH and moisture content of purchased pharmaceutical samples. A mixture of methanol and hydrochloric acid (0.01N Methanolic HCl) was used to determine the biochemical properties of doxycycline hyclate. UV detector set at 349 nm was used to monitor the effluent. The purified water was used as solvent. Results: In 1% aqueous solution of doxycycline, three samples (4th, 5th and 7th) showed lower pH values of 1.97, 1.98, and 1.99 respectively. Furthermore, the same samples indicated the additional moisture contents of 2.81%, 2.85% and 2.83% respectively while considering the acceptance level (1.4% to 2.8%). The method proved to be linear (R2=0.993), precise (RSD=0.79% for inter-day precision), accurate (Recovery=100.59%) and selective regarding possible impurities and excipients of the samples. The doxycycline content obtained in the sample analysis was within the range of 84.05% to 85.80%. Conclusion: The optimized and validated method may be successfully employed to perform routine quality control analyses. Investigation of the pH, moisture content and potency of doxycycline hyclate in different samples give a general view of local pharmacies trade and ensure that the method applied here was validated for this kind of analysis.