The feasibility of applying instrumental epithermal neutron activation analysis for the determination of the thickness of thin layers of gold, chromium and nickel, sputtered on glass tubes, was investigated experimentally. The method is based on the irradiation of thin slices of glass tubes after the sputtering process, and measurement of the radioactivity derived from 198Au, 51Cr and 58Co radionuclides which were used to determine the thickness of gold, chromium and nickel layers, respectively. Under the experimental conditions of the sputtering system, the coating thickness of gold, chromium and nickel were found to be 0.34 ± 0.01, 0.034 ± 0.001 and 0.044 ± 0.001 μm respectively. The method is selective, accurate, highly sensitive and may be routinely applied on a laboratory scale.