A simple and reliable HPLC-UV method for Kaempferol (KPF) determination in a Kaempferol-loaded nanoemulsion (KPF-NE), samples from mucosa permeation/retention studies, and murine brain was developed and validated according to international guidelines. The analyses were performed on a reversed-phase C18 column at 35 °C and under UV detection at 368 nm. The mobile phase was composed of methanol:formic acid 0.1% (75:25, v/v) and was eluted at an isocratic flow rate of 1.0 mL/min. The method was selective and sensitive for KPF analysis in matrix extracts, and linear in the range of 0.25–7.5 μg/mL. The method was also considered precise, accurate, and robust. The recovery rates of KPF from the porcine nasal mucosa and murine brain were higher than 85%. Low matrix effect was observed to determine KPF, including biological matrices. The applicability of the method was confirmed in all different approaches, i.e., quantification of KPF in nanoemulsion, in vitro permeation/retention of KPF across porcine nasal mucosa, and in vivo quantification of KPF in brain samples after nasal administration in rats. Thus, the method is effective and reliable to determine KPF in different real samples. The proposed method, therefore, provides a useful quantification approach to routine processes, to the development of drug delivery systems, and to KPF quantification in different biological matrices. Furthermore, the method is applicable in bioavailability studies and the developed formulation (KPF-NE) is suitable for preclinical trials in different brain disorders.