Speaking at an economic forum in St Petersburg, Mr. Putin strongly reaffirmed Russia's view that the Syrian president's forces were not responsible for the attack on April 4 in the opposition-controlled town of Khan Sheikhoun.
He said the attack was a provocation intended to put the blame on the Syrian ruler, insisting: "Assad didn't use those weapons."
The US blamed Mr. Assad's government for the attack which killed at least 90 people, and launched nearly 60 cruise missiles at the Shayrat air base in the central province of Homs, where it claims the attack originated, AP reported.
The UN said a team from the international chemical weapons watchdog found exposure "to sarin or a sarin-like substance" in samples from the attack on the town in Idlib province.
Mr. Putin said Moscow had offered the US and its allies the chance to inspect the Syrian base for traces of the chemical agent and criticized them for refusing to do so.