From 1960 he trained at the Institute of Organic Chemistry and Biochemistry (IOCB) of the Czechoslovak Academy of Sciences in Prague and had been a researcher there since 1963.

Since 1976 he had collaborated on the development of new antiretroviral drugs with Erik De Clercq of the Rega Institute for Medical Research at the Catholic University of Leuven, Belgium.

[8] His key contribution is the synthesis of nucleotide analogues, synthetic mimetics of the building blocks of RNA and DNA, that have found utility as inhibitors of viral replication.

[9] Although ineffective as originally synthesized by Holy because of impermeability conferred by the negative charged phosphonate, conjugation with POM and POC pro-drug moieties developed by David Farquhar at M.D.

Anderson Cancer Center, has made these nucleotide analogues into clinically effective drugs for the treatment of Hepatitis B (biPOM-Adefovir, Hepsera), and biPOC-Tenofovir Tenofovir.