While the I, Claudius ones are all great, none of them quite beats Livia’s comment to Tiberius following Augustus’s death - ‘By the way, don’t touch the figs.’ Brilliant punchline to the most remarkable scene in the whole series.
This may be a little long for a “single greatest line”, but the I, Claudius references brought this to my mind:
Senator: There are those who say you cannot hear properly, you cannot speak properly, and that you’ve got no experience of government
Claudius: And that I am besides half-witted. Senators, it is true that I am hard of hearing, but you will find it is not for want of listening. As for speaking, again, it’s true I have an impediment. But isn’t what a man says more important than how long he takes to say it? It’s true again I have little experience of government. But then, have you more? I at least have lived with the imperial family who has ruled this empire ever since you so spinelessly handed it over to us. I’ve observed it working more closely than any of you. Is your experience better than that? As for being half-witted, well, what can I say - except that I have survived to middle age with *half* my wits, while thousands have died with *all* of theirs intact. Evidently, *quality* of wits is more important than *quantity*. Senators, I shall do nothing unconstitutional; I shall appear at the next session of the senate where you may confirm me in my position or not as you wish. But if it pleases you not to, explain your reasons to them [points at the Praetorians]