This study covers the quantification of the covalent attachment of gelatin type B (GelB) and the subsequent adsorption of Fibronectin (Fn) on poly-ε-caprolactone (PCL) surfaces, functionalised with 2-aminoethyl methacrylate (AEMA) by means of post-plasma UV-irradiation grafting. As typical surface characterisation tools do not allow quantification of deposited amounts of GelB or Fn, radiolabeled analogues were used for direct measurement of the amount of immobilized material. Bolton-Hunter GelB (BHG) and Fn were radioiodinated with (131)I and (125)I respectively and S-Hynic GelB (SHG) was labeled with (99m)Tc. Immobilisation of (131)I-BHG or (99m)Tc-SHG on both PCL and PCL-AEMA scaffolds was performed in analogy with earlier work. SPECT images on scaffolds coated with (99m)Tc-SHG conjugates were acquired on a U-SPECT II camera. There was a clear difference in the amount of deposited (131)I-BHG between blanco and AEMA-grafted PCL on 2D samples. No significant differences in immobilization behaviour were observed between (99m)Tc-SHG and (131)I-BHG. Subsequent immobilisation of Fn was successful and depended on the amounts of deposited GelB. SPECT imaging on cylindrical 3D scaffolds confirmed these findings and showed that the amount of immobilized (99m)Tc-SHG was depth dependant. The architecture of the scaffolds strongly influences the distribution of GelB within these structures. Furthermore, there is a clear difference in the homogeneity of the protein coating when different GelB immobilization protocols were applied. This study shows that radiolabeled compounds are a rapid and accurate tool in the quantitative and qualitative evaluation of the biofunctionalisation of AEMA grafted PCL scaffolds.