Purpose: This study aimed to examine the degradation of tigecycline in Mueller Hinton broth (ca-MHB), as knowledge about bacterial susceptibility is key for therapeutic decisions. Methods: Antioxidative stabilizers were evaluated on tigecycline stability in a quantitative chromatography assay and tigecycline induced kill against Staphylococcus aureus (ATCC29213) was determined in time kill studies. Results: Ascorbic acid caused rapid degradation of tigecycline and resulted in loss of antibacterial activity. Tigecycline was stabilized in aged broth by 2% pyruvate and bacterial growth, and tigecycline killing was similar to fresh broth without supplementation, but independent of age. Conclusion: Our results underline the importance of using freshly prepared ca-MHB or the need for stabilizers for tigecycline susceptibility testing while using aged ca-MHB.