An artificial spawning substrate for brook trout (Salvelinus fontinalis Mitchill) was developed for use in laboratory bioassay studies involving reproduction. The substrate consisted of a glass spawning box containing a compartmented screen egg retainer on top of which was placed a coarse screen with attached gravel to simulate the stream bottom. Egg viability from wild and hatchery trout spawning on this substrate averaged 96%. This new substrate design reduces considerably the time required to determine daily spawning activity in laboratory brook trout experiments.