Prelogs main interest was focused on alkaloids. He found an ideal topic in the elucidation of the structure of solanine; he continued his work on Cinchona alkaloids and started to investigate strychnine. He showed that Robert Robinsons formula for strychnine was not correct. Although the formula he proposed was also not the right one, the discovery increased his international prestige. Later he worked on elucidating the structures of aromatic Erythrina alkaloids with Derek Barton, Oskar Jeger and Robert Burns Woodward.
At mid-century, the instrumental revolution necessitated a new approach to structural elucidation. Purely chemical methods had become outdated and had lost some of their intellectual appeal. Recognizing the growing importance of microbial metabolites, Prelog started working on these compounds, which possess unusual structures and interesting biological properties. It led him into antibiotics, and he subsequently elucidated the structures of such compounds as nonactin, boromycin, and rifamycins. For Prelog, natural products represented more than a chemical challenge. He considered them a record of billions of years of evolution.
In 1944 at the ETH, Prelog managed to separate enantiomers with "asymmetric" trivalent nitrogen by column chromatography at a time when this method was still in its infancy. His work on medium-sized alicyclic and heterocyclic rings established him as a pioneer in stereochemistry and conformational theory and brought an invitation to give the first Centenary Lecture of the Chemical Society in London in 1949. He synthesised medium-sized ring compounds with 8 to 12 members from dicarboxylic acid esters by acyloin condensation and explained their unusual chemical reactivity by a "nonclassical" strain because of energetically unfavorable conformations. He also contributed to the understanding of Bredt's rule by showing that a double bond may occur at the bridgehead if the ring is large enough.
In his research of asymmetric syntheses, Prelog studied enantioselective reactions and established rules for the relationship between configuration of educts and products. From Prelog's researches into the stereospecificity of microbiological reductions of alicyclic ketones and the enzymic oxidation of alcohols, he contributed not only to the knowledge of the mechanism of stereospecificity of enzymic reactions in general but also to the structure of the active site of the enzyme.
Specifying the growing number of stereoisomers of organic compounds became for Prelog one of his important aims. In 1954 he joined R. S. Cahn and Christopher Ingold in their efforts to build a system for specifying a particular stereoisomers by simple and unambiguous descriptors that could be easily assigned and deciphered: The CIP system (Cahn-Ingold-Prelog) was developed for defining absolute configuration using "sequence rules". Together they published two papers. After Cahn and Ingold died, Prelog published a third paper on the topic. In 1959, Prelog obtained Swiss citizenship.