Nuclear and G-protein coupled receptors are considered major targets for drug discovery. FXR and GP-BAR1, two bile acid-activated receptors, have gained increasing consideration as druggable receptors. Because endogenous bile acids often target both receptor families, the development of selective ligands has been proven difficult exposing patients to side-effects linked to an unwanted activation of one of the two receptors. In the present study we describe a novel library of semi-synthetic bile acid derivatives obtained by modifications on the cholane scaffold. The pharmacological characterization of this library led to the discovery of 7-hydroxy-5-cholan-24-sulfate (7), 6-ethyl-37-dihydroxy-5β-cholan-24-ol (EUDCOH, 26) and 6-ethyl-37-dihydroxy-24-nor-5β-cholan-23-ol (NorECDCOH, 30) as novel ligands for FXR and GP-BAR1 that might hold utility in the treatment of FXR and GP-BAR1 mediated disorders.