The four have been held since Thursday despite having immunity under ICC protections and a United Nations Security Council resolution.
"These four international civil servants have immunity when on an official ICC mission," ICC President Judge Sang-Hyun Song said in a written statement. "I call on the Libyan authorities to immediately take all necessary measures to ensure their safety and security and to liberate them."
Gadhafi faces charges in the ICC relating to his father's crackdown on pro-democracy protesters who eventually toppled the regime. The ICC staff members are attached to the Office of Public Counsel for the Defense, which has been appointed by the court to act as his defense.
The four were in Libya to meet with Gadhafi about possibly retaining his own lawyers. The ICC said the visit had been cleared ahead of time with the new Libyan government.