Resonant laser ionization and mass separation of ²²⁵Ac

²²⁵Ac is a radio-isotope that can be linked to biological vector molecules to treat certain distributed cancers using targeted alpha therapy. However, developing ²²⁵Ac-labelled radiopharmaceuticals remains a challenge due to the supply shortage of pure ²²⁵Ac itself. Several techniques to obtain pure ²²⁵Ac are being investigated, amongst which is the high-energy proton spallation of thorium or uranium combined with resonant laser ionization and mass separation. As a proof-of-principle, we perform off-line resonant ionization mass spectrometry on two samples of ²²⁵Ac, each with a known activity, in different chemical environments. We report overall operational collection efficiencies of 10.1(2)% and 9.9(8)% for the cases in which the ²²⁵Ac was deposited on a rhenium surface and a ThO ₂ mimic target matrix respectively. The bottleneck of the technique was the laser ionization efficiency, which was deduced to be 15.1(6)%.