Objectives: The aim of this research is to optimize the isolation of PBMCs in order to maximize the yield and purity of the preparations Study design: This clinical observational study is part of a wider project. The goal of that project is to optimize the validation of candidates for non-small cell lung cancer tumor markers. Materials and methods: The study involved 40 subjects (healthy volunteers) who were divided into 4 groups to examine different conditions of isolate preparation. Blood was sampled in two tubes; for PBMC isolation (a commercial Ficoll gradient tube) and for PBMC isolation performance evaluation (a K3-EDTA tube). The examined parameters were the centrifugation length and the relative centrifugal force. Optimal conditions for the isolation of PBMC are determined by counting cells in a hematology counter. Results: Centrifugation speed does not affect the number of cells isolated and their utilization, while centrifugation time significantly affects the total number of leukocytes, lymphocytes and monocytes. Also, centrifugation time affects on % of isolated cells of leukocytes, lymphocytes and monocytes. The length and speed of the centrifugation do not affect the platelet count and % of platelets isolated. Conclusion: Mononuclear cells isolation is optimized at 30 minutes of centrifugation at 1600 RCF because under these conditions the highest utilization of PBMCs was obtained and the contamination of the mononuclear cell isolate by other blood fractions was reduced.