Due to the indubitable role of vitamins in maintaining human health, a lot of attention has been paid to the methods and analytical procedures of their determination. Voltammetric methods are of particular interest as they do not involve complex sample preparation, however, close values of the redox potential of some vitamins may result in peak overlapping, hindering the quantitative analysis. This paper addresses the separation of overlapped reduction peak of vitamins B1 and B3 by means of Continuous Wavelet Transform (with appropriately selected mother wavelets) and differentiation of the recorded voltammograms. These numerical algorithms allowed to obtained linear, single-variable calibration functions that comply with the applied in analytical chemistry criteria regarding the correlation coefficients and the limit of detection and quantification. Their relatively simple implementation does not include any manual interpretation step; thus, these algorithms provide impartial and reproducible results. The proposed methodology has been applied in the determination of vitamins B1 and B3 in dietary supplements, providing results consistent with the declaration of the manufacturer.Graphic abstract