The present work demonstrates the implementation of aptamers as capture molecules for a wide range of target classes in lateral flow assay applications. The targets were chosen in order to cover a wide range of target classes (small sized - metabolite, medium sized - protein, and large sized - whole cell/spore). For each target class one target molecule was selected as representative and appropriate aptamers were used for lateral flow assay development. The work points out that the implementation of aptamers as capture molecules in a universal lateral flow test platform was successful independent form target size. Furthermore, the limit of detection for p-aminohippuric acid in urine (200 ppm), lysozyme in white wine (20 ppm), and Alicyclobacillus spores in buffered orange juice (>8 CFU/mL) were determined using aptamers as capture molecules. The whole approach is considered as a proof of concept, regarding the ability of aptamers as an alternative to antibodies (in conjunction with directive 2010/63/EU on the protection of animals used for scientific purposes) in lateral flow applications. 2017 Elsevier Inc. (C) All rights reserved.