In 2010, Mexico celebrates 200 years since the beginning of the Independence war that gave rise to the independent Mexican Empire in 1821, and afterwards to the Mexican Republic. This document had two original copies; one of them was lost in a fire at the beginning of twentieth century, while the second was stolen and finally returned to Mexico in 1960, after a long history of events. This document is kept in the General Archives of Nation (AGN), Mexico.The “Independence Act of the Mexican Empire of 1821” was written on paper using iron-gall inks. The document has two parts: a declaration and a set of 36 signatures of Iturbide and other people involved in establishing the Independence of Mexico.The non-destructive study of this document was carried out in order to answer several questions: legitimacy, composition of the materials (paper and inks), deterioration conditions and a possible sequence of writing and the signatures. For these purposes several in situ techniques were used: optical microscopy, ultraviolet and infrared light imaging, portable X-ray Fluorescence and Raman Spectroscopy. This work presents the main results of this analytical methodology applied to the Mexican Independence Act. The results indicate that several inks were used in the manuscript and that the paper has an aging consistent with a nineteenth century document. From these results, we consider that the document examined is genuine and not a copy or facsimile of the original act.