The full validity of an analytical method requires a good awareness of the tools available, and is therefore a major asset in guiding laboratories towards the adoption of appropriate approaches, not only to enable both analytical validation and estimation of measurement uncertainty, but also to make less effort and experience. Indeed, a graphical approach namely uncertainty profile intended to assess the validity and estimate the measurement uncertainty of analytical procedure has been applied, its success reside to the construction of the β-content, γ-confidence tolerance interval. The purpose of this paper is to describe and test a full validation of an LC-MS method for the determination of nevirapine in human plasma using the uncertainty profile as a new holistic validation approach. By exploiting data collected under intermediate precision conditions, we have come up with a good assessment of the uncertainty of chemical measurements through an innovative formula, based on the β-content tolerance interval without referring to any extra effort and additional experiments. Three statistical procedures, namely: Satterthwaite approximation, the generalized pivotal confidence method and the modified large simple procedure have been used to build this tolerance interval. Regardless of the statistical model tested, the proposed analytical procedures were validated over the selected validation domains since 66.7% of the future results falls inside the acceptance limits of ± 15%.