Tigecycline (TIGE) is the newest tetracycline derivative antibiotic with low toxicity, it is used for management of infectious diseases caused by Gram‐positive and Gram‐negative bacteria. Hence, an efficient, selective and sensitive method was developed for analysis of TIGE in commercial formulations, human plasma and urine. The spectrofluorimetric technique based on the reaction of secondary amine moiety in TIGE with 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in slightly alkaline medium producing a highly fluorescent product measured at 540 nm (λex at 470 nm) after heating for 15 min at 75°C. The proposed strategy was upgraded and approved by ICH rules and bio‐analytical validated using US‐FDA recommendations. A linear relationship between fluorescence intensity and TIGE concentration was observed over the concentration range 40–500 ng mL−1 with limit of quantification (LOQ) 21.09 ng mL−1 and limit of detection (LOD) 6.96 ng mL−1.The ultra‐affectability and high selectivity of the proposed strategy permits analysis of TIGE in dosage form, human plasma and urine samples with good recovery ranged from 97.23% to 98.72% and from 99.36% to 99.80% respectively, without any interfering from matrix components. Also, the developed strategy was used to examine the stability of TIGE in human plasma and applied for pharmacokinetic investigation of TIGE.