Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-effective treatments of various infectious diseases as they may increase drug bioavailability, control drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0 ± 11.8 nm and Zeta Potential −42.5 ± 1.5 mV suggestive of important stability. DSC/TGA and FT-IR assessments suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were developed and validated according to regulatory agencies in order to fast and assertively determine AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability studies. The quantification method for AmB in NLC suspension presented linearity in 5.05–60.60 μg mL−1 range (y = 0.07659x + 0.05344) and for AmB in receptor solution presented linearity in 0.15–10.00 μg mL−1 range (y = 54609x + 263.1), both with r ≥ 0.999. EE% was approximately 100% and according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46 h. In the meantime, a micellar AmB solution demonstrated an instability pattern after 7 h of contact with the medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a prominent degradation of AmB (raw-material), with 20.3 ± 3.5% at the first hour, reaching 43.3 ± 7.0% after 7 h of study. Next, particles faster disruption in acid environment was evidenced by measuring the NLC size variation by DLS and by the loss of the bluish sheen, characteristic of the nanostructured system macroscopically observed. Finally, safety studies depicted that NLC-AmB presented reduced toxicity in fibroblast cells, corroborating with AmB aggregated form study. Therefore, an innovative AmB formulation was fully characterized and it is a new proposal for in vivo investigations.