In an internal calibration, a compound (called internal standard) will be added to each standard solution. In this way, the standard solutions contain the compounds to be dosed, at ascending concentrations, as well as an internal standard added at the same concentration at each calibration point. This internal standard is added to all test samples at the same concentration.

This calibration is thus more complicated to undertake than external calibration - in practice, an internal calibration is performed when the analytical method lacks repeatability or reproducibility. A typical example of application is gas chromatography, where the injected volumes are hard to reproduce.