In the current study, a rapid and sensitive LC-QTOF-MS/MS method for the determination of brinzolamide in dried blood spots (DBS) was developed and validated. This novel sample collection, storage and transfer technique was suitable for analyzing a drug with high distribution into red blood cells and negligible plasma levels. The method included an isocratic mobile phase consisting of methanol and 10 mM ammonium formate (90:10, v/v) and detection in positive electrospray mode (ESI+). The flow rate was adjusted to 0.350 mL/min yielding retention times of 1.7 min for both brinzolamide and internal standard (IS) rabeprazole on a Cyano analytical column, respectively. The validation of the proposed method over the concentration range 0.500–20.0 μg/mL was performed in compliance with EMEA and FDA guidelines, assessing all major performance characteristics. Inter- and intra- assay precisions were less than 14%, while inter- and intra- assay accuracies varied from 92.2 to 111%. No matrix effect was observed and the mean brinzolamide extraction recovery was 93.5%. The method was successfully applied to real DBS samples from patients in steady state condition, receiving brinzolamide ophthalmic suspension 1% (w/v) for several months. Initial concentrations were corrected due to hematocrit effect, using image processing algorithm written in Matlab.