Isotope separation is the process of concentrating specific isotopes of a chemical element by removing other isotopes. The use of the nuclides produced is varied. The largest variety is used in research (e.g. in chemistry where atoms of "marker" nuclide are used to figure out reaction mechanisms). By tonnage, separating natural uranium into enriched uranium and depleted uranium is the largest application. This process is crucial in the manufacture of uranium fuel for nuclear power plants and is also required for the creation of uranium-based nuclear weapons (unless uranium-233 is used). Plutonium-based weapons use plutonium produced in a nuclear reactor, which must be operated in such a way as to produce plutonium already of suitable isotopic mix or grade.

While chemical elements can be purified through chemical processes, isotopes of the same element have nearly identical chemical properties which makes this type of separation impractical, except for separation of deuterium.

There are three types of isotope separation techniques:

The third type of separation is still experimental; practical separation techniques all depend in some way on the atomic mass. It is therefore generally easier to separate isotopes with a larger relative mass difference. For example, deuterium has twice the mass of ordinary (light) hydrogen and it is generally easier to purify it than to separate uranium-235 from the more common uranium-238. On the other extreme, separation of fissile plutonium-239 from the common impurity plutonium-240, while desirable in that it would allow the creation of gun-type fission weapons from plutonium, is generally agreed to be impractical.

All large-scale isotope separation schemes employ a number of similar stages which produce successively higher concentrations of the desired isotope. Each stage enriches the product of the previous step further before being sent to the next stage. Similarly, the tailings from each stage are returned to the previous stage for further processing. This creates a sequential enriching system called a cascade. There are two important factors that characterize the performance of a cascade. The first is the separation factor, which is a number greater than 1. The second is the number of required stages to get the desired purity.

To date, large-scale commercial isotope separation of only three elements has occurred. In each case, the rarer of the two most common isotopes of an element has been concentrated for use in nuclear technology:

Some isotopically purified elements are used in smaller quantities for specialist applications, especially in the semiconductor industry, where purified silicon is used to improve crystal structure and thermal conductivity, and carbon with greater isotopic purity to make diamonds with greater thermal conductivity.

Isotope separation is an important process for both peaceful and military nuclear technology, and therefore the capability that a nation has for isotope separation is of extreme interest to the intelligence community.