A method is described which allows to determine the content of the so-called C(80) tetra-acid molecules (TA) in calcium naphthenate deposits. The method consists of four steps. Molecules present in the deposit are dissolved in a mixture of toluene and 2-butanol after an acidic treatment. All acid molecules are then selectively extracted and concentrated by a solid-phase extraction (SPE) method. After derivatization of acids into their naphthacyl esters to increase the sensitivity of the detection, TA is separated and detected by reversed-phase HPLC with UV detection. We have checked that all the steps are quantitative and the method appears selective. The TA content can be determined in presence of other naphthenic acids. Using this methodology we have determined the TA content in three calcium naphthenate deposits from different oil fields. It appears that these deposits have a similar TA concentration between 28 and 41% (w/w).