The invention relates to a new approach for the analysis of pyro-sequencing signals. It comprises a method for analyzing pyrosequencing signals. The invention permits to identify and/or quantify each unique nucleotide sequence contribution in a global pyro-sequencing signal. The invention also relates to the creation of a dictionary of standardized pyro-sequencing signals for its use within the method. The invention also relates to the determination of a nucleotide dispensation order that enhances the analysis of a pyro-sequencing signal by the method.