Myeloid derived suppressor cells (MDSC) is a heterogeneous subset of immature and mature cells of the myeloid lineage, undergoing expansion during pathologic conditions, and able to perform strong immune suppressive functions. It has been shown that cryopreservation selectively impacts the polimorphonuclear (PMN) MDSC viability and recovery, and alters the correct analysis of MDSC subsets. In laboratory practice, cryopreservation is often inevitable, in particular in multicenter studies where samples have to be shipped to a centralized laboratory. Aim of the present work was to set out a new protocol to evaluate the frequency of PMN-MDSC in thawed cells by flow-cytometry. PBMC were isolated from HIV+ patients and healthy donors, and were cryopreserved for at least ten days. After thawing, two different protocols were used: 1. standard protocol (SP) consisting of staining with the antibodies mix and then fixing with formalin 1%; 2. thawed protocol (TP) in which fixation foregoes the staining with the antibodies mix. Results showed that PMN-MDSC frequency in ex vivo PBMC evaluated by means TP was comparable to that analysed by SP, indicating that the protocol did not alter PMN-MDSC quantification in ex vivo cells. We then demonstrated that PMN-MDSC frequency in thawed PBMC tested by TP was almost identical to the frequency obtained in ex vivo cells evaluated by using SP. However, we observed that after three hours of culture post-thawing, PMN-MDSC were not assessable anymore with both SP and TP. In conclusion, we herein demonstrated that fixing PBMC soon after thawing and before antibody staining allows preservation of PMN-MDSC integrity and a reliable cells quantification. Thus, it is possible to phenotipically identify PMN-MDSC in cryopreserved PBMC, consenting adequate test precision and accuracy as well as making multicentre research more feasible.