House dust mite allergens Der p 1, Der p 2, and Der p 23 are recognized as major clinically relevant allergens worldwide; however, it is difficult to obtain these proteins in purified form from a natural source, which limits their use in molecular targeted immunotherapy and in vivo diagnosis. In this study, we developed and validated robust methodologies for the large-scale purification and individual characterization of native nDer p 1, nDer p 2, and nDer p 23 allergens from the natural sensitization source, Dermatophagoides pteronyssinus. Each allergen was isolated through an independent downstream process based on successive chromatographic steps, achieving high purity and preserving the structural integrity. Molecular standardization was performed in vivo in 27 mite-allergic patients by skin prick testing (SPT), enabling the separate determination of histamine equivalent potency (HEP) values: 7.43 µg/mL for nDer p 1, 8.11 µg/mL for nDer p 2, and 1.55 µg/mL for nDer p 23. These data establish a direct relationship between the protein concentration and biological activity for each major allergen. In conclusion, the successful production and biological standardization of native nDer p 1, nDer p 2, and nDer p 23 proteins provide well-defined reagents for in vivo molecular diagnosis and enable more precise and reproducible standardization compared with complex allergen extracts.