Super-resolution optical imaging techniques rely on validation standards. In this regard, it is of paramount importance to develop consistent and reliable well-defined reference samples. In this study, we employ a DNA origami scaffold to engineer a multicolor quantum dot hybrid nanostructure, and evaluate it through a recently proposed quantum dot-based spectral separation technique. Our findings underscore the utility of multivalent DNA structures, which serve as a robust and precise scaffold for the nanoscale placement of quantum dots. The employed spectral resolution method offers a straightforward and rapid means of imaging acquisition, compatible with standard confocal or fluorescence microscopes possessing spectral signal separation capabilities and a single excitation laser wavelength. This combined methodology represents a promising avenue for advancing super-resolution optical imaging techniques.