A highly sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for quantification of the propafenone (PF), 5-hydroxypropafenone (5-OHPF) and N-depropylpropafenone (N-DPF) on human dried blood spot (DBS). The assay procedure involves a solid-liquid extraction of PF, 5-OHPF and N-DPF and amlodipine (internal standard, I.S.) from dried human DBS cards using water and acetonitrile. The chromatographic resolution was achieved on a BEH C18 column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile with 0.1% formic acid at flow rate of 0.6mL/min. The UHPLC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Total run time of analysis was 1.1min and elution of PF, 5-OHPF, N-DPF and I.S. occurred at 0.69, 0.6, 0.68 and 0.73min, respectively. A detailed method validation was performed as per the regulatory guidelines and the standard curves found to be linear in the range of 5.11-1000ng/mL for PF and 5-OHPF and 0.51-100ng/mL for N-DPF with a correlation coefficient of ≥0.99 for all the drugs. The intra- and inter-day accuracies were in the range of 95.6-107 and 93.5-103; 93.4-106 and 96.3-107 and 87.9-103 and 96.5-102%, for PF, 5-OHPF and N-DPF, respectively. The intra- and inter-day precisions were in the range of 2.50-5.52 and 3.38-5.18; 2.16-6.34 and 3.23-4.94 and 2.63-7.55 and 1.56-10.2%, for PF, 5-OHPF and N-DPF, respectively. The validated assay method was successfully applied to a pharmacokinetic study in humans. The key pharmacokinetic parameters AUC0-∞ and Cmax were 6057±1526, 2002±515 and 525±202 ng*h/mL and 653±183, 295±37.5 and 68.4±13.6ng/mL for PF, 5-OHPF and N-DPF, respectively.