A new calibration strategy for elemental bioimaging based on online isotope dilution analysis (IDA) and laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) was developed and applied for the quantification of platinum in rat kidney tissues. A dry 194Pt spike aerosol was added in a post-cell setup, and the natural 194Pt/195Pt isotope ratio of the sample aerosol from laser ablation was changed accordingly. Spike mass flow determination was carried out based on reversed IDA using a reference standard. Quantitative data obtained by the new approach correlated well with those obtained by external calibration when analyzing parallel tissue slices of rat kidney from cisplatin perfusion studies. The novel quantification approach is traceable to SI units, as IDA is an definitive method. Signal drifts are compensated as the second isotope acts as an internal standard.