Otolith microchemistry is a widely used technique for elucidating life history patterns in fishes. This typically involves sectioning the otolith and collecting elemental signatures via laser ablation. But this requires time-intensive handling that may influence results. As an alternative to traditional cut/polish/ablate techniques, we tested depth-profiling laser ablation, which offers reduced handling and contamination risk. To validate depth-profiling as a robust method for collecting trace element otolith microchemistry data, we constructed composite otoliths using otolith materials from fishes of different origins (freshwater, seawater). Test ablations were conducted on composite diadromous otoliths at a range of spot sizes and pit depths. We measured tailing and fractionation effects in the following elements; Na, Mg, K, Mn, Zn, Rb, Sr, and Ba. Given appropriate instrument parameters, depth-profiling can accurately collect elemental concentration data both between and within top and bottom layers of an otolith composite across a range of spot sizes and pit depths. Analytical power and lag effects were dependent on spot size, highlighting the importance of optimizing spot size based on sample morphology and instrument parameters. ; The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author.