The specific details depend on the particular plate reader. Some plate readers allow you to select any wavelength for excitation and emission. Others require specific filter sets for the wavelengths needed. Some plate readers require selecting a gain setting and a focal height, or going through an optimization procedure. Others do not.

The concentration of the fluorescent dye should be in the nanomolar range in most cases. For very bright fluorophores, it may be sufficient to use a single-digit nanomolar concentration. For less bright ones, double-digit nanomolar may be needed. The gain setting, if there is one, will be influenced by the fluorophore concentration, with higher concentrations requiring lower gain.

Use a solid black plate for fluorescence measurement and choose top reading, if there is a choice.

Because fluorescent dyes are hydrophobic, they tend to stick to plastic plates. This can be avoided by including in the solution a low concentration of a nonionic detergent, such as 0.01% Triton X-100. The detergent concentration should be below the concentration at which the detergent forms micelles (critical micellar concentration, CMC).

For fluorescent dyes with ionizable groups (amines, carboxylic acids), use a buffer to set the pH of the solution to one that is conducive to the fluorophore being at its brightest.