Aim: A new one-step flow cytometry procedure has been recently demonstrated for identifying subjects with infections, but only for fresh whole blood samples. The goal of this study was to assess its applicability on frozen samples, by proposing a new method to perform the sample freezing directly and easily. Methods: Fresh blood was tested, then frozen either directly or with dimethylsulfoxide and serum. Common markers of white blood cells as well as infection-related biomarkers were tested. Results: All percentages of leucocyte subsets and levels of infection-related biomarkers were significantly correlated between frozen and fresh samples. Conclusion: The direct freezing method enables an accurate assessment of common cellular sub-populations and of levels of important infectious biomarkers via flow cytometry.