Cyclophilin A (CypA) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A (CsA). Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template. Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands. Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans. Small but effective: We describe the design and synthesis of small-molecule inhibitors for cyclophilin A, based upon a dimedone template. Mass spectrometry was used as an initial screen to quantify the affinity of the ligands for the protein. The biological role of cyclophilins in the nematode Caenorhabditis elegans was investigated with active inhibitors and fluorescent derivatives as chemical probes. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.