The absorbance of UV and visible (UV–visible) radiations is measured using a UV–visible spectrophotometer. Deuterium lamps, which emit UV radiation, and tungsten lamps, which produce radiation in the visible region, are used as light sources in UV–visible spectrophotometers. A monochromator or prism is used for diffraction grating, i.e., to split the incoming radiation into different wavelengths. A system of slits is used to focus the desired wavelength on the sample cell. Samples for UV–visible spectrophotometer must be in the liquid phase. Therefore, solid organic compounds should be dissolved in a suitable solvent prior to analysis. The sample is then placed in a sample holder known as a cuvette. Depending on the sample, and the radiation used, the cuvette may be made of quartz crystal, glass, or plastic. Glass or plastic sample cells are used in visible spectroscopy because their spectral cutoffs are around 350 nm—meaning they effectively transmit visible light but absorb UV. UV spectroscopy uses quartz cells with a lower cutoff of around 200 nm to allow transmission of UV light.

Since the solvent will also absorb light, a sample blank of the solvent alone is used prior to analyzing the actual sample. Selecting a suitable solvent for ultraviolet spectroscopy is critical for accurate absorbance measurements. Water, 95% ethanol, and hexane are the most commonly used solvents in UV–visible spectroscopy. A suitable solvent also helps get the fine structure of an absorption band. Nonpolar solvents do not form hydrogen bonds with the solute. As a result, the final spectrum of the solute will be similar to the spectrum observed in the gaseous phase, where fine structure is often present. In contrast, polar solvents do form hydrogen bonds with the solutes, which means that the fine structure of an absorption band will not be present.

A photomultiplier tube, charge-coupled devices, and photodiode or photodiode array are usually used as detectors. The intensity of the light passed through the sample cell is referred to as I. In a single-beam instrument, all of the light passes through the sample cell. In a double-beam instrument, the light is split into two beams; one beam is used as the reference, and the other beam is passed through the sample. In modern diode-array spectrophotometers, photodiode detectors are used to record the entire spectrum at once.