The IEDDA reactivity of bis-hydroxyaryl-Tz 4-6 was first studied in comparison to 1 (DMT), 2 (PA2), and 3 (2Pyr2) by stopped-flow spectrophotometry. Second-order rate constants (k2) for the click reaction with the PEGylated rTCO-derivative 8 (rTCO-PEG4) were determined in acetonitrile (MeCN) at 25 °C and phosphate buffered saline (PBS, pH 7.4) at 37 °C (Fig. ). This allowed us to compare reactivities across the entire series, despite the insufficient solubility of 2 in MeCN and of 4 in aqueous media. Considering both datasets, tetrazines 1, 2, and 4 show only limited click reactivity, while significantly higher rate constants were observed for the two bis-hydroxypyridyl-Tz 5 and 6. The IEDDA reactivity of Tz 5 in MeCN (51 M -1 s -1 ) is only slightly lower compared to that of bis-pyridyl-Tz 3 (62 M -1 s -1 ). Despite a more pronounced difference in PBS (with 3 showing an approx. 6-fold higher reactivity than 5), a rate constant of 1500 M -1 s -1 was observed for the reaction of rTCO-carbamate 8 with Tz 5 (Fig. ). Therefore, comparing Tz capable of near-complete release, h2P2 (5) reacts more than 120-fold faster than the Tz-diacid 2 (PA2).