Prelog was born in Sarajevo, Bosnia and Herzegovina, at that time within the Austro-Hungarian Empire, and moved to Zagreb, Croatia with his parents at the outbreak of World War I. Educated in the capital of Croatia, he graduated from the Czech Institute of Technology in Prague in 1929, receiving a degree as a chemical engineer.
At the beginning of the Great Depression, he started to work in a private plant laboratory in Prague, in charge of the production of rare chemicals that were not available on the market at that time. In 1935, he was invited to join the Technical Faculty of the University of Zagreb, where he took the post of lecturer in organic chemistry. He also taught students of chemical engineering.
The same year he started his research works in the pharmaceutical factory "Kaštel" (currently Pliva). In 1941, when the German troops occupied Zagreb, he and his wife accepted the invitation of his colleagues, and fled to Switzerland. Prelog continued his work at the Swiss Federal Institute of Technology (ETH). In 1957 he was promoted, and took the position of director of the organic laboratory, previously filled by Leopold Ružička.
Based on the formal findings and general rules, his studies focused on stereochemistry. Prelog examined aspects of chirality in chemistry, and introduced the so-called R–S system into organic chemistry. Working with stereoisomers, he created a new version of isomerisation, which he called cyclostereoisomerisation. Prelog purified the chemical adamantine from petroleum whose structure of carbon atoms is diamond shaped.
He was a keen traveller and gave lectures on his research findings in plenty of places all over the world. He was an excellent lecturer. Prelog was awarded the 1975 Nobel Prize in chemistry for his research into the stereochemistry of organic molecules and reactions. He formulated the Cahn–Ingold–Prelog priority rules, which are used in organic chemistry to name the stereoisomers of a molecule.