The diagnosis of Lesch-Nyhan syndrome is based initially on the distinctive pattern of the child's symptoms, most commonly involuntary muscle movements or failure to crawl and walk at the usual ages. In some cases the first symptom is related to overproduction of uric acid; the parents notice “orange sand” in the child's diapers. The “sand” is actually crystals of uric acid tinged with blood.

Measuring the amount of uric acid in a person's blood or urine can not definitively diagnose Lesch-Nyhan syndrome. It is diagnosed by measuring the activity of the HPRT enzyme through a blood test. When the activity of the enzyme is very low it is diagnostic of Lesch-Nyhan syndrome. It can also be diagnosed by DNA testing. This is also a blood test. DNA testing checks for changes (mutations) in the HPRT gene. Results from DNA testing are helpful in confirming the diagnosis and also when the child's family is interested in prenatal testing for future pregnancies.

Prenatal diagnosis is possible by DNA testing of fetal tissue drawn by amniocentesis or chorionic villus sampling (CVS). Fetuses should be tested if the mother is a carrier of a change (mutation) in her HPRT gene. A woman is at risk of being a carrier if she has a son with Lesch-Nyhan syndrome or someone in her family has Lesch-Nyhan syndrome. Any woman at risk of being a carrier should have DNA testing through a blood test.