In this work, the reliability of Dried Blood Spot (DBS) as a sampling technique for drug analysis was studied by Ultra High Performance Liquid Chromatography coupled to Photodiode-Array and Fluorescence Detection (UHPLC-PDA-FLUO). DBS microsampling, a technique based on placing a drop of blood in a cotton support that is allowed to air dry, has lately noticed an increase in use in bioanalysis. Even thought it offers several advantages compared to common blood sampling methods, it also shows some limitations for quantitative analysis due to the dependence on different factors. In this study, the influence of some of them (haematocrit, blood volume and sampling position) has been investigated, using amiloride, propranolol and valsartan drugs as model compounds. According to the results, it has been concluded that the sampling position and the haematocrit have influence in the accuracy and precision of the quantitative results, therefore limiting the use of this technique. On the other hand, dispersion of the analytes in the blood drop depends on their physicochemical properties which implies that the distribution of each analyte must be carefully studied during method development.