Etoposide is a topoisomerase II enzyme inhibitor type chemotherapeutic agent which is widely used in the therapy of various cancers. Its short half-life and toxicity to normal tissues are the major drawbacks in its clinical applications. Polymeric nanoparticulate drug delivery systems are rational carriers to deliver etoposide with higher efficiency and fewer side effects. In addition tubular shaped drug carriers are found to show a great potential for drug delivery on the basis of promising results regarding particle shape and cellular uptake. In this study, etoposide loaded polymeric tubular nanocarriers have been developed by template wetting method using porous anodic aluminum oxide membranes as templates. The developed poly(methyl methacrylate) nanocarriers were evaluated for structural analysis, in vitro drug release studies and drug release kinetics. Accurate and reliable determination of the drug release from newly developed nanocarriers, is of great importance. For this reason a selective and sensitive reversed phase liquid chromatography method was developed and fully validated from the point of system suitability, specificity, linearity and range, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and robustness for the reliable determination of etoposide. Stability indicating capability was shown with forced degradation studies and the chromatographic conditions were optimized on ACE 5C18 (150 mm × 4.6 mm I.D., 5 μm) analytical column. Related to the calibration results ETP was found linear in the range between 0.2 from 100 μg mL−1 with the LOD as 0.015 μg.mL−1. The resultant conditions were applied for the selective and sensitive determination of etoposide from its commercial dosage form with the high accuracy values (99.82–100.65%). The method was successfully detected assay of etoposide release from newly developed polymeric tubular nanocarriers, which was found as 72.2% at the end of 24 h.