A new high-performance liquid chromatographic method coupled with UV detection (HPLC-UV) to quantify ethambutol (ETH) post permeation studies following microneedle administration has been developed. This method involves the derivatization of ETH with phenethyl isocyanate (PEIC) at room temperature for 90 min. The separation of the derivative was performed using a C18 column that utilised a mobile phase consisting of 25 mM sodium dihydrogen phosphate buffer (with 1% v/v triethylamine, pH 3.0 adjusted using orthophosphoric acid) and methanol (25 : 75 v/v). The developed analytical method was validated according to the standards set by the International Council on Harmonization (ICH) guidelines. The method is linear for drug concentrations within the range of 0.39-12.5 μg mL ( = 0.9999). The validated method was found to be specific, precise, and accurate. Moreover, the ETH derivative was found to be stable under specific storage conditions. In addition, a simple and straightforward extraction procedure for extracting and quantifying ETH from the skin was developed and evaluated. The extraction procedure displayed recovery rates that range from 101.77 ± 7.10% to 102.33 ± 8.69% indicating high extraction efficiency. The developed method was utilised in assessing the permeation of ETH across dermatomed neonatal porcine skin following microneedle application. Collectively, the simple and stable HPLC method developed in this study may be of great utility in screening formulations for ethambutol within a preclinical setting through permeation studies.