Not the all-time hardest but...
The first time I fought the second level's boss in Gradius V. After a long level, and a barrage of power-ups, ya get to the boss. So I fight him, and I win, and I think, well that wasn't so tough. But wait. He has a second form. Oh. Well, after that one, which gets a little crazy at times, I pull through. Ok then, pretty chall-wha? A third form. This one I recognize from Gradius III. So I fight it, end up losing my options and what not, but barely win on my last life, but finally, I've triumph-...DAMMIT! A fourth form?!? The hell? And then I get destroyed, in a big big way. Oy.
It's one crazy fight.