On-bead screening of one-bead one compound (OBOC) libraries is an ultra fast surface based primary high-throughput screening (HTS) method. Typically the binding of a tagged target protein to bead immobilized compounds or its altered enzymatic activity are detected. For an efficient and reliable ligand discovery process secondary assays to confirm on-bead compound activity in homogeneous solution are key to exclude artifacts and weak binders. Ideally they should allow to flag hit compounds with undesirable biophysical properties such as aggregation, unspecific binding, or insufficient solubility and the like. Here we demonstrate that miniaturized and parallelized equilibrium dialysis is an excellent and generic secondary confirmation method for hit compounds identified by on-bead screening. We further show that microscale dialysis can be reliably performed prior to decoding and resynthesis even with hit-compounds cleaved from the single beads. Down-scaling of the method takes advantage of the fluorescent tag, AIDA, which is integrated as permanent tracer in our library design. Our results suggest that microscale equilibrium dialysis followed by high performance liquid chromatography (HPLC) analysis is a generic, cheap, and meaningful confirmation method for identifying the most promising candidates within a series hit compounds derived from fluorescently tagged one-bead one-compound libraries.