Objective: The aim of this study is to identify possible diagnostic applications of intracellular markers: LDH, NSE and ferritin from the cell lysate for peripher blood mononuclear cells (PBMC) optimization procedure. Study design: Clinical observational study Materials and Methods: The study included 40 healthy subjects of both sex. Approximately 8 mL of venous blood was sampled to each subject in 3 vacutainers. Individual blood samples were divided into 3 tubes from which isolates were individually isolated and then lysed peripheral mononuclear and verified isolation and lysis of peripheral mononuclear isolates. Results: Optimal lysis criteria, considering arithmetic mean and standard deviation, for lysate protein concentrations and activity, LDH and total lysate proteins: F: -20 ° C, T: + 37 ° C, 2 cycles; ferritin: F: -70 ° C, T: + 37 ° C, 2 cycles; NSE: F: 20 ° C, T: + 37 ° C; 1 cycle. A protein ratio lysate to serum alters optimal lysis conditions for ferritin: F: -20 ° C, T: + 37 ° C; 1 cycle, whereas remaining parameters keep the same values. After processing data by ANOVA, only the lysis temperature condition is statistically significant. Freezing at -20 °C is suitable for LDH, NSE and total proteins, while no criteria are suitable for ferritin. Conclusion: The optimal freezing point for cell lysis procedure, at which the greatest utilization of biochemical parameters is achieved: NSE, LDH and total lysate proteins is -20 °C and for lysate ferritin is -70 ° C. Ferritin value in lysate/serum ratio does not show significant temperature condition. The number of F/T cycles is statistically insignificant for each of the parameters used in the study.