An optical system is provided for achieving enhanced rejection of scattered excitation light and superior signal-to-noise performance when reading microplate wells. The optical system uses an axial configuration in which the excitation beam incident upon the sample propagates along the axis of the microplate well. Excitation light from a light source, such as a lamp or fiber optic bundle, is collimated into a beam using a lens. A reflective pick-off mirror is then used to reflect the collimated excitation beam upward along the well axis. A focusing lens, with a diameter exceeding the diameter of the collimated excitation beam, is used to focus the excitation beam in the well. The same broad lens is used to collimate the emitted fluorescent light, of which a large percentage propagates axially past the pick-off mirror towards a second focusing lens that focuses the emission beam onto the face of a fiber optic bundle. The emitted light is later filtered and detected using at a position that is optically shielded from the aforementioned optical system. The optical system is incorporated into a microplate reader or automated assay instrument in order to provide a compact assembly for sensitive fluorescence measurements either above or below the microplate. The optical system further enables the simultaneous measurement of absorbance and fluorescence in a compact optical configuration.