Busulfan (Bu) is currently used as a part of the conditioning regimen for hematopoietic stem cell transplantation (HSCT).Therapeutic drug monitoring (TDM) of Bu is usually performed in plasma. The Dried Blood Spot (DBS) sampling technique is a rapid, requires lower volumes of blood and less invasive method compared to the conventional plasma method to perform the TDM of Bu, especially in children. The objective of the study was to evaluate the possibility of clinical implementation of DBS method for individual Bu dose adjustment. The impact of hematocrit on the DBS assay performance was also investigated. DBS and DPS (Dry Plasma Spot) were prepared simultaneously from the venous blood collected from healthy volunteers and was spiked with Bu calibrators, and analyzed using a validated LC-MS/MS technique. Clinical samples from 15 pediatric patients who received Bu, collected at different time points were used to prepare DBS and DPS. Theoretical plasma Bu concentrations were calculated from DBS concentrations considering patient's specific hematocrit and fraction of drug bound to plasma proteins. Bland-Altman analysis was used to define the agreement between the two methods considering a predefined limits of agreement of 20%. Analysis of clinical samples indicated a good correlation between DBS and DPS (R2=0.862 and a slope of 0.88), as well as between DPS and theoretical plasma levels of Bu estimated from DBS (R2=0.868 and a slope of 0.92). For the in vitro analysis a good agreement was shown between DBS and DPS, with an average difference of -5.4 % and 95% limits of agreement (LoA) 4.6% to -15.3%. However, for the clinical samples, DBS method did not meet the clinical acceptance limits, indicating an average difference of 10.7% ( 95% LoA from -20.7 to 42.1%) between DPS and DBS, and an average difference of 7.5% ( 95% LoA from -22.9% to 37.9%) when DPS was compared to theoretical plasma concentrations obtained from DBS. Changes in hematocrit levels did not have any significant influence on Bu measured concentrations in both in vitro and in vivo studies (R2=0.01, slope of 0.05 and R2=0.009, slope of 0.39, respectively).These results show that although DBS sample collection is a useful method for the TDM of Bu, further studies are needed before it can be implemented in clinical practice.