In the current study, β-cyclodextrin (β-CD) inclusion concurrent with cationic surfactant shielding was firstly evaluated for the successful stabilization of L-ascorbic acid (AA) under ambient lab conditions followed by ultra-high performance liquid chromatography (UHPLC) and online preconcentration capillary electrophoresis (CE). The combination of 0.44 mM β-CD with 10 mM cetyltrimethyl ammonium bromide (CTAB) cationic surfactant (β-CD-CTAB) was significantly reduced the degradation of AA at pH 7.0 with 11 d stability. However, the combination of β-CD with sodium dodecyl sulfate (SDS) anionic surfactant (β-CD-SDS) gave only 1 d stability of AA. Moreover, β-CD-CTAB complex was stabilized AA with 175- and 12-fold enhancement compared with individual β-CD and CTAB, respectively. The formation of AA-β-CD-CTAB ternary complex was proved by following the chromatographic and electrophoretic behavior of AA in the absence and presence of β-CD and CTAB species. Thus, the obtained results were very promising and clearly provided a higher stability for AA without any molecular changes which normally limited the application of different analytical methods. After that, the reversed phase UHPLC/UV and CE/UV methods were developed for the quantitative determination of AA in different commercially available samples including pharmaceuticals, drinking juices and milk samples. Good linearity within the concentration range 1–1000 µg/mL was achieved with coefficient of determinations (r2) higher than 0.9995. The limits of detection were 0.39 and 0.56 µg/mL by UHPLC and CE, respectively. The proposed methods could be suitable for the quality control of AA, providing simple, rapid and reliable approaches for routine analysis of different pharmaceuticals and food.