Once the membrane has been incubated with the second antibody and the excess has been washed away, a detection substrate is added, which reacts with the conjugate of the secondary antibody and produces a measurable result. Chemiluminescence or colorimetric detection are two commonly used methods for this.

In chemiluminescence, a substrate is added which produces light-emission when it reacts with the catalysing conjugate of the second antibody. The light-emission can be detected on a photographic film or by a digital camera.

Colorimetric detection is instead based on that a substrate is added which is converted by the conjugate to a certain coloured (and thereby visible) product.

In both cases, the amount of protein can be measured by densiometry (i.e. measuring the amount of light-emission/colour of the band). Thereby, both a qualitative and quantitative analysis of a protein can be done by Western blotting.