The conversion of ceramide into sphingomyelin (SM) was only recently considered in relation to cancer. Overexpression of the CERT protein, responsible for a highly specific inter-organelle ceramide transfer step along the de novo SM synthesis pathway, has been associated with multi-drug resistance of cancer cells. Identification of new CERT antagonists may therefore lead to potential resensitizing agents. This work describes the first attempt to design a ceramide-based fluorescent probe optimised to evaluate the binding of potential CERT ligands. A prototypical structure with an ω-labelled sphingosine backbone was selected. Its possible recognition mode by CERT was first evaluated by means of a precursory molecular modelling study. Three derivatives with various amide chain lengths were prepared and tested. The binding efficiency was shown to be proportional to the lipophilicity of the acyl moiety. The best compound bearing a C16 amide fragment was used to implement a practical binding experiment. Facile assessment of the recognition by the CERT START domain of various structures was thus ensured. Metabolism and imaging experiments were also used to illustrate the capacity of the proposed fluorescent ceramide analogue to mimic the natural ceramide cellular behaviour. This work led to the synthesis and evaluation as an efficient CERT START domain ligand of a ω-biotinylated ceramide, a potential probe to develop the screening of new CERT antagonists.