Many stocking programs are performed without any subsequent assessments to determine the survival of thestocked fish, often due to the difficulties in distinguishing stocked from wild fish. A series of optimisation trials wereundertaken to determine the suitability of calcein (2,4-bis-[N,N0'di(carbomethyl)-aminomethyl]fluorescein) for chemicallymarking hatchery-reared Australian bass, Macquaria novemaculeata (Steindachner), fry and fingerlings. The technique wasoptimised by trialling immersion times for salt (2.5 or 5 min), calcein concentrations (0.5 or 1%) and calcein immersion times(5 or 10 min). A general-purpose modulated probe fluorometer was used as a non-lethal detection method to establish markretention and distinguish between calcein-marked and unmarked fish. Five minutes of saline immersion, followed by 10 minof calcein immersion at a calcein concentration of 1% was the optimal marking method. Growth and survival of fish was notaffected as a result of any of the calcein-marking methods. It is therefore effective to calcein-mark Australian bass fry andfingerlings before stocking and to non-lethally detect marked individuals using a field-portable meter to assist in theassessment of stocking programs. Findings from this study may also reflect the potential use of calcein to chemically mark fryand fingerlings of other fish species.