Cucurbit[n]uril (CB[n]) macrocycles (especially CB[5] to CB[8]) have shown exceptional attributes since their discovery in 2000. Their stability, water solubility, responsiveness to several stimuli, and remarkable binding properties have enabled a growing number of biological applications. Yet, soon after their discovery, the challenge of their functionalization was set. Nevertheless, after more than two decades, a myriad of CB[n] derivatives has been described, many of them used in cells or in vivo for advanced applications. This perspective summarizes key advances of this burgeoning field and points to the next opportunities and remaining challenges to fully express the potential of these fascinating macrocycles in biology and biomedical sciences.