Objectives: In this work we presented a highly sensitive, selective and validated method for determination of tedizolid phosphate (TEDP) antibiotic, based on its native fluorescence in aqueous solution. Methods: The maximum fluorescence intensity was measured at 408 nm after excitation at 298 nm after optimization of all experimental conditions. Results: The measured fluorescence was directly proportional to the concentration of the drug over the range of 2-30 ng/mL with a limit of detection of 0.13 and limit of quantification of 0.44 ng/mL The method succeeded to determine TEDP in its pharmaceutical dosage form and in human plasma with mean % recovery of 100.49 ±1.32 and 99.40 ± 2.18 respectively. Conclusion: The developed data was found to be with a good agreement with a valid method. The method was validated according to ICH guidelines for determination of the drug in its pure form and dosage form and according to FDA Guidance for Industry, Bioanalytical Method Validation for determination of TEDP in human plasma.