Asparaginase is an enzyme used for the treatment of hematopoietic diseases such as acute lymfoblastic leukaemia and non-Hodgkin’s lymphoma. The use of asparaginase as antileukemic agent is based on direct enzymatic activity on circulating asparagine, an essencial aminoacid for tumoural cells as opposed to healthy ones which capable of synthethizing it. The aim of the present work was to purify recombinant periplasmatic asparaginase produced by the methylotrophic yeast Pichia pastoris, expressing the ASP3 gene from Saccharomyces cerevisiae. Cellular extract, obtained by previously described methodology, was submitted to ammonium sulphate precipitation fractionation (0-40%, 40-80% and 80-100% saturation). The 40-80% saturation fraction presented the highest enzymatic specific activity which was measured by the detection, at 550 nm, of a compound resulting from hydroxilamine reaction. Further analysis by SDS-PAGE under reducing conditions revealed two major bands with molecular masses of 42 and 44 kDa. The 40-80% fraction was also submitted to size exclusion chromatography on a Superdex 200 HR 10/30 equilibrated and eluted with 50mM Tris-Cl pH 7.2 150mM NaCl buffer. Chromatographic separation profile revealed two main peaks, SPase I and SPase II, with estimated molecular masses of 85 and 47 kDa respectively. Enzymatic activity was detected only for SPase I fraction and SDS-PAGE under reducing conditions revealed two major bands of 42 and 44 kDa. These bands were excised, submitted to trypsin digestion and the extracted peptides were analyzed by liquid chromatography coupled to electrospray ionization-ion trap mass spectrometry (LC-ESI-ION TRAP MS).  Aminoacid sequences obtained from MS/MS spectra were submitted to nrNCBI database and both bands were identified as asparaginase II coded by ASP3 from Saccharomyces cerevisiae. Hence, our data indicate that we are on the edge of obtaining both homogeneous and enzymatically active recombinant enzyme and that this cloned asparaginase is being expressed as a mixture of homodimeric and/or heterodimeric molecules, although the nature (different post-transcritional and/or post-translational processing) of this heterogeneity remains to be determined.