"They stealthily and cowardly assassinated Gen. Soleimani in a terrorist fashion. This resulted in a disgrace for America,” the Leader told a huge crowd in Tehran.
Ayatollah Khamenei said Iran's missile response to the assassination dealt a blow to the American prestige.
The missile attack was a "military blow, but more important it was a blow to the American grandeur", the Leader said.
Ayatollah Khamenei said American "clowns" who are claiming to be standing with the Iranian people are lying.
He also hit out at British, German and French leaders who admitted this week to have launched under pressure by US President Donald Trump a process that could lead to European sanctions being reimposed on Iran.
Ayatollah Khamenei called them "footmen in the real sense of the word", telling them "You are too small to bring the Iranian nation to its knees."
The Leader said the European states "cannot be trusted", adding their engagement in negotiations is always associated with "deceit and fraud".
He also touched on the "bitter" tragedy of the loss of lives in the accidental downing of a Ukrainian airliner outside Tehran, saying necessary measures must be taken in order to avoid a repetition of the incident.
"The plane crash was a bitter accident, it burned through our heart," Ayatollah Khamenei said. "But some tried to... portray it in a way to forget the great martyrdom and sacrifice" of Major Soleimani, the Leader said.
For the first time in eight years, Ayatollah Khamenei is leading Friday prayers in Tehran’s Imam Khomeini Grand Mosque, where huge crowds of worshipers have already gathered.
Massive protest rallies are also expected to be held after the prayers in condemnation of the United States which recently assassinated the Middle East's most prominent anti-terror commander General Qassem Soleimani.