Prelog attended elementary school in Sarajevo, but in 1915, as a child, Prelog moved to Zagreb (then part of the Austro-Hungary) with his parents. In Zagreb he graduated from elementary school. At first, he attended gymnasium in Zagreb, but soon afterwards, his father got a job in Osijek, so he continued his education there. He spent two years in Osijek gymnasium, where he became interested in chemistry under the influence of his professor Ivan Kuria.
In 1922, as a 16-year-old boy, his first scientific work was published in the German scientific journal Chemiker Zeitung. The article concerned an analytical instrument used in chemical labs. Prelog completed his high school education in Zagreb in 1924. Following his fathers wishes, he moved to Prague, where he received his diploma in chemical engineering from the Czech Technical University in 1928. He received his Sc.D in 1929. His teacher was Emil Votoček, while his assistant and mentor Rudolf Lukeš introduced him to the world of organic chemistry.
Upon leaving the Czech Technical University, Prelog worked in the plant laboratory of the private firm of G.J. Dríza in Prague; few academic positions were available due to the Great Depression. Prelog was in charge of the production of rare chemicals that were not commercially available at that time. He worked for Driza from 1929 until 1935. During the time, he got his first doctoral candidate, a company owner at Driza. He performed research in his spare time, investigating alkaloids in cacao bark.