Dopamine receptors, which belong to the family of G protein-coupled receptors, are very substantial regulators in the brain and therefore important targets in drug discovery. Radioligand binding assay has been the method of choice for screening novel dopaminergic drugs for decades. We demonstrate that fluorescent ligand BodipyFL-SKF83566 binding to dopamine D1 receptors expressed in budded baculovirus particles can be characterized with fluorescent anisotropy (FA) based assay and that this is a valuable alternative to the radioligand binding assay. High binding affinity (KD = 5.2 nM) and fast association and dissociation kinetics (half-lives 40 s and 70 s, respectively) make BodipyFL-SKF83566 a suitable fluorescent ligand for this type of experiments. Good correlation between pKi values of 11 different dopaminergic ligands determined in competition binding experiments with radioligand [3H]SCH23390 or BodipyFL-SKF83566 (R2 = 0.96, slope not significantly different from unity) further validates the FA assay. In addition to competitor's affinity, the method also enables to quantify the apparent pIC50 values in time and hence kinetic properties of an unlabeled ligand can be estimated from the same competition binding experiment. Due to the homogenous nature of the FA assay reactions can be monitored in real time without any risk of precipitation of receptors in budded baculovirus particles. Despite the lack of coupled G proteins, the proposed novel assay system allows on-line monitoring of ligand binding to dopamine D1 receptors that could be easily applicable in early drug screening.