Substances with a low molecular weight (< 900 Da) are classified as small molecules and are ubiquitous in nature and in the human body. Small molecules can be found in the environment where they are produced as secondary metabolites by a variety of fungi, bacteria, and plants. In the human body, small molecules also play important roles in metabolism, such as signal transduction and regulatory processes. Since small molecules are ubiquitous in nature as well as in the human body, an increased interest in monitoring these substances in the diagnostic environment has emerged in the last decades. Detection of the small molecule 25-hydroxy vitamin D3 (25(OH)D) has been extensively explored in the literature. 25(OH)D is the main biomarker of metabolic vitamin D supply. Vitamin D insufficiency has been associated with an increased risk of cancer and bone disease. Current detection methods of this molecule, using high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and immunoassays, are time consuming, expensive, and require trained personnel. In this work, a novel detection approach using a highly specific aptamer directed against 25(OH)D was established based on the Target-Induced Dissociation (TID) method. Dissociation of a complementary, labeled oligonucleotide upon target binding by the aptamer results ina signal change proportional to the analyte. First, the 25(OH)D-binding aptamer was studied in terms of structural properties as well as binding affinity. Then, complementary oligonucleotides were designed based on the aptamer structure. The oligonucleotides were evaluated in MicroScale Thermophoresis (MST) and microarray experiments to identify the optimal oligonucleotide sequence for maximized displacement. Further microarray experiments were then performed to immobilize the aptamer on a surface and verify whether the immobilization process affected the binding functionality of the aptamer. Finally, the novel TID assay was characterized in terms of limit of detection (LOD), limit of quantitation (LOQ), and sensitivity. The assay for 25(OH)D detection successfully detected the small molecule with a LOD of 32 nM in buffer. LOQ was determined at 109 nM in aptamer binding buffer.