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In a letter to the White House, Sen. Tim Kaine of Virginia and two dozen other Democratic senators said the Trump administration has provided "no legitimate legal justification" for the strike.
Senators are demanding more information from the administration on the situation and use of U.S. military power.
"Our armed forces are not law enforcement agencies," said Sen. Jack Reed of Rhode Island, the top Democrat on the Senate Armed Services Committee, in a floor speech this week.
"They are not empowered to hunt down suspected criminals and kill them without trial," Reed said.
The Trump administration has claimed self-defence as a legal justification for the strike, with Secretary of State Marco Rubio arguing the drug cartels "pose an immediate threat" to the nation. The U.S., which has designated Tren de Aragua as a terrorist organization, has indicated more military strikes on drug targets could be coming as it seeks to "wage war" on cartels.