Seif's transfer to face charges relating to the bloody repression of the 2011 uprising that toppled his father however is a moot point as he is being held by a Libyan militia rather than any central authority in the chaos-wracked country.
The presiding ICC judge Erkki Kourulas struck down four grounds of appeal before the world's war crimes court, saying "in the present case the Appeals Chamber confirms the (pre-trial chamber's) decision and dismisses the appeal."
This included Tripoli's inability to transfer Seif, Gaddafi one-time heir apparent, to the Libyan capital from his prison in the hilltop stronghold of Zintan, where he is currently being held by militia members.
Tripoli appealed the original decision a few days later, in June.
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Seif, 41, and Gaddafi's former spy chief Abdullah Senussi, around 64, have been charged for their roles in violent attempts to put down the 2011 uprising in the desert country that eventually toppled Kadhafi's regime.
His court-appointed lawyer was unable to attend the hearing, so the trial was adjourned to May 25 to allow him to help his client.
"The ICC Appeals decision today reinforces Libya's long overdue obligation to surrender Seif Gaddafi to The Hague for fair trial," Human Rights Watch's international justice director Richard Dicker said.
"If Libya refuses, the international community should step up and demand his surrender," said Stephanie Barbour, head of Amnesty International's Centre for International Justice in The Hague.
Militias are blamed for growing unrest in the North African country since the 2011 NATO-backed uprising that killed dictator Gaddafi after more than 40 years in power.