<jats:title>Abstract</jats:title><jats:p>A selective and sensitive method has been developed for the analysis of free and bound forms of <jats:italic>O</jats:italic>‐phosphoamino acids, such as <jats:italic>O</jats:italic>‐phosphoserine, <jats:italic>O</jats:italic>‐phosphothreonine and <jats:italic>O</jats:italic>‐phosphotyrosine, in urine samples by gas chromatography (GC). For free <jats:italic>O</jats:italic>‐phosphoamino acid analysis, the urine sample was extracted with trichloroacetic acid and run through an ion exchange column. For total (free plus bound) <jats:italic>O</jats:italic>‐phosphoamino acid analysis, the urine sample was hydrolysed in acid and base in order to release the <jats:italic>O</jats:italic>‐phosphoamino acid from peptides or proteins. <jats:italic>O</jats:italic>‐Phosphoamino acids in these prepared samples were converted into their <jats:italic>N</jats:italic>‐isobutoxycarbonyl trimethyl ester derivatives and then measured by GC with flame photometric detection (FPD‐GC). The calibration curve was linear in the range of 10–500 ng for each <jats:italic>O</jats:italic>‐phosphoamino acid, and the detection limits were 30–80 pg as injection amounts. <jats:italic>O</jats:italic>‐Phosphoamino acids in the urine samples could be selectively determined by the FPD‐GC method without any influence from coexisting substances. The recoveries of <jats:italic>O</jats:italic>‐phosphoamino acids added to urines and urine hydrolysates were 90–98% and relative standard deviations were 1.5–8.0%. By using this method, we investigated an age dependence of <jats:italic>O</jats:italic>‐phosphoamino acid excretion in normal subjects.