The US Anti-Doping Agency announced the move after Armstrong's former team-mate Tyler Hamilton last year claimed he used the banned performance-enhancing substance EPO while preparing for the 2001 Tour.
Armstrong, who has always denied that he took performance-enhancing drugs during his career, is alleged to have engaged others in a massive doping conspiracy from 1998-2011.
The group, which includes trainer Pepi Marti of Switzerland and doctors Pedro Celaya of Luxembourg and Luis Garcia del Moral of Spain, face competition bans if found guilty.
The American, who survived cancer and battled back to health to win cycling's greatest event, has been accused of breaking doping laws in the past, but has always protested his innocence.
The agency claimed Armstrong used EPO, along with blood transfusions, testosterone, corticosteroids and masking agents.
Last night, his lawyer Robert Luskin said the latest claims were baseless.
Mr Luskin said: "These charges are a product of malice and spite, and not evidence."