Biallelic pathogenic variants in CYP27A1 causing cerebrotendinous xanthomatosis (CTX) lead to disruption of both the neutral and the acidic pathways for conversion of cholesterol to the primary bile acids, the taurine and glycine conjugates of chenodeoxycholic acid (CDCA) and cholic acid (CA). This in turn leads to a loss of the bile acid feedback inhibition of the rate-limiting step in the neutral pathway, cholesterol 7α-hydroxylase, exacerbating the build-up of neutral pathway intermediates such as 7α-hydroxy-cholest-4-en-3-one, 7α,12α-dihydroxy-cholest-4-en-3-one and 5β-cholestane-3α,7α,12α-triol. 7α-Hydroxy-cholest-4-en-3-one can be converted to cholestanol which, with cholesterol, builds up in the brain, atheromatous plaques and xanthomata. 5β-Cholestane-3α,7α,12α-triol can undergo C25 hydroxylation, leading to the build-up of 5β-cholestane-3α,7α,12α,25-tetrol glucuronide in plasma and urinary excretion of a range of 5β-cholestane-pentol glucuronides (including 22,25-,23,25- and 24,25-pentols). As a result of these metabolic changes, a variety of biochemical tests can be used to detect and monitor efficacy of treatment of CTX. These include plasma cholestanol (measured alongside other sterols/stanols), plasma 7α-hydroxy-cholest-4-en-3-one, 7α,12α-dihydroxy-cholest-4-en-3-one, blood/plasma 5β-cholestane-3α,7α,12α,25-tetrol glucuronide and urine bile alcohol glucuronides (often measured alongside urinary bile acids). The relative merits of these tests in the setting of a chemical pathology laboratory are discussed with a consideration of the ways in which CTX may present.