Despite the growing interest in circulating cell-free DNA (ccfDNA) analysis in various clinical fields, especially oncology and prenatal diagnosis, few studies on sample handling have been reported and no analytical consensus is available. The lack of consistency between the various protocols for sample handling and the techniques used for ccfDNA analysis is one of the major obstacles in translating ccfDNA analysis to clinical practice. Although this point is highlighted regularly in the published reviews on ccfDNA analysis, no standard operating procedure currently exists despite several ongoing clinical studies on ccfDNA analysis. This review examines the preanalytical parameters potentially affecting ccfDNA concentration and fragmentation at each preanalytical step from blood drawing to the storage of ccfDNA extracts. Analysis of data in the literature and our own observations revealed the influence of preanalytical factors on ccfDNA analysis. Based on these data, we determined the optimal preanalytical protocols for ccfDNA analysis and ultimately, a guideline for the translation of ccfDNA analysis in routine clinical practice.