Condemning the Russian president's actions abroad and repression at home, Biden said facing down Putin was "critical to checking further aggression down the road."
"We're looking for him to, in our view, act more rationally and if he does not, we will continue to confront what I characterize as pure aggression," he said in a speech at a Washington think tank.
In March 2014, Russia annexed Ukraine's Crimea region after a pro-European government came to power in Kiev.
Biden urged European leaders meeting in June to keep sanctions on Russia in place until the terms of a ceasefire agreement are met.
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"Sanctions on Russia must and will remain in place," he said, adding that allies must be ready to mete out more sanctions if "Russia again moves beyond the line of contact."
He said Putin was "at his core" practical, "he will push as far as he can ... Until he reaches a resistance."
"President Putin is wagering that he has greater staying power," Biden added. "In Ukraine, he's betting that he can outlast the current reformist pro-European government and undermine it economically."
Some believe that providing even defensive weapons to Kiev would escalate a conflict against Russia that Ukraine has no chance of winning.
Biden also framed the crisis as a test for democracies, with far reaching implications around the world.
"What happens in Ukraine and how the West and the world respond has, I think, consequential implications for the nature of the international order," Biden said at the Brookings Institution.
"China and many other nations are watching very closely how the world responds."