¹Department of Pharmaceutical Chemistry, College of Pharmaceutical Sciences, Karnataka, Bengaluru-560078, India DOI : 10.29228/jrp.140 A simple and sensitive high-performance liquid chromatographic (HPLC) method is established to separate and quantify the related substance present in two alkylating agents. The use of the traditional approach of analytical validation, in practice or in the literature, is communal. However, statistical verification, that looks separately the two errors (such as absolute bias and repeatability) to make a decision, presents a risk to declare that an analytical method is valid while it is not, or equally. To minimize this peril, a new approach based on the concept of total error was proposed. In this paper, we reveal the applicability and simplicity of the both methods based on the total error approach: accuracy profile and uncertainty profile. Proposed study demonstrated by validation case of a liquid chromatographic LC method for the quantification of related compounds present in two alkylating agents. Both methods showed good linearity response (> 0.995) with repeatability (%relative standard deviation less than 2%) and accuracy (94 to 106%). Accuracy profile was found within the range of ±10% and risk profile ±5% between the two series respectively. The excellence of the total error approach was presented since it enables successfully to validate the analytical procedure as well the calculation of the measurement uncertainty at each concentration level. Keywords : Related substances; Quantification; Total error; HPLC; Validation