Guidelines for pharmaceutical recombinant proteins are very rigid and structural protein characterization in all batches is necessary. For this purpose, the most used technique is peptide mapping by reversed phase chromatography (RP-HPLC) after protein enzymatic hydrolysis. Comparing retention times between sample and standard peptides, "errors" in the protein structure can be identified. The common defects presented by those engineered proteins are: sequence deletions and insertions, wrong disulfide bridges, asparagine deamination and metionine oxidation. The problem of RP-HPLC is the relative low sensitivity sometimes not compatible with peptide detection. In this work, we have developed a new physical chemical methodology for structural characterization of recombinant human erithropoietin (rh-EPO) from their formulations, using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Initially, different rh-EPO formulations were fractionated by RP-HPLC. The chromatographic fractions corresponding to this protein were recovered and submitted to hydrolysis with trypsin and the peptides formed were analyzed by MALDI-TOF spectrometry. Different matrices, spectrometer operational conditions and sample preparations were tested. Best results were achieved with sinapinic acid in linear acquisition mode. More than 60% of rh-EPO sequence was identified in the mass spectra. In all samples, disulfide bridges were present in the correct positions. In one sample, an O-glycosylation site was partially non glycosylated. These results suggest that MALDI-TOF methodology can be used in samples with amounts equal or below 20 µg of rh-EPO that are incompatible with peptide screening by classical reverse phase chromatography