Background: The dried blood spot (DBS) technique is a convenient alternative sample collection method to venipuncture. The use of DBS was fi rst widely introduced in the 1960's by Dr. Guthrie to measure levels of phenylalanine in newborns for the diagnosis of phenylketonuria. The scientifi c community has recently expressed interest in applying the DBS technique for quantifi cation of drugs in biological fl uids. Materials and Methods: Reversed phase high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for the determination of hydrochlorothiazide (HCTZ) in human plasma and DBS samples were developed and validated. Furthermore, clinical samples collected from human volunteers as a part of pharmacokinetic (PK) study and PK data from plasma were compared against DBS. Results: The range of reliable response in plasma and DBS was 2-200 ng/mL with a correlation coeffi cient ≥0.997. Inter-day accuracy was always within 85-115%, and inter-day precision for plasma and DBS were <15%. Conclusions: Comparison of HCTZ PK data in DBS and plasma yielded good correlation, indicating that DBS can be used as a plasma alternative for PK analyses in vivo.