Adsorption isotherm is the most fundamental information related to chromatography. To calculate the parameters of Langmuir adsorption isotherm of thymidine, frontal analysis (FA) and elution-curve method (ECM) were adopted in reversed-phase high performance liguid chromatography (RP-HPLC). In FA, the concentration of stationary phase was measured from the elution curves and the isotherm was determined by regression analysis, while the parameters by ECM were obtained by parameter optimization. The adsorption isotherms of thymidine from the two methods were very similar. The superiority of ECM over FA was that the consumption of sample was less and only one or two injections of sample were required.