To assess Tz stability, we incubated 100 µM solutions of the bispyridyl-Tz 3, 5 and 6, and the aryl-H-Tz 15-17 in PBS at 37 °C and monitored the degradation by serial HPLC measurements (see Supporting Information). The hydroxylated 4-pyridyl-Tz 6 showed the lowest stability of the symmetric tetrazines with a halflife (t1/2) of less than 2 hours. In agreement with our recent findings regarding the chemistry of phenyl-and pyridyl-Tz, we observed that bis-hydroxypyridyl-Tz 5 exhibits higher stability (t1/2 ~5 hours) despite its increased click reactivity, although it is less stable than Tz 3 (t1/2 ~13 hours). In contrast, while the hydroxypyridyl-H-Tz 17 degraded rapidly in PBS (t1/2 < 2 hours), the hydroxyphenyl-H-Tz 16 showed the highest stability (t1/2 ~45 hours) within the series, even surpassing its non-hydroxylated analog 15 (t1/2 ~34 hours). Based on these results and due to the higher click reactivity as well as release performance of 5 and 16 (compared to 6 and 17, respectively), these tetrazines were used in further experiments to demonstrate bioorthogonal application.