Background/Objectives: Ya Pra Sa Kra Tung Pew Pai Nok is a traditional Thai medicinal formulation historically used for fever and inflammatory skin symptoms. This study characterized a lactose-containing vacuum-dried extract, evaluated its in vitro anti-inflammatory activity, and incorporated it into a topical spray formulation. Methods: The formulation comprised Brachypterum scandens (Roxb.) Miq., Tiliacora triandra (Colebr.) Diels., and Tamarindus indica L. in equal proportions. Genistein-7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside (GTG) was selected as a chemical marker and quantified using a validated HPLC-DAD method. Results: The method showed good selectivity and linearity over 4–20 µg/mL, with intra- and inter-day precision RSD values of 4.04% and 3.43%, recoveries of 97.41–103.65%, and LOD and LOQ values of 0.15 and 0.49 µg/mL, respectively. CCK-8 analysis showed that both vacuum-dried extract preparations, with and without lactose, maintained RAW264.7 cell viability above 80% across all tested concentrations. At non-cytotoxic concentrations, the lactose-containing extract significantly reduced nitric oxide and prostaglandin E2 production in LPS-stimulated RAW264.7 macrophages, demonstrating in vitro anti-inflammatory activity of the extract. A topical spray containing 5% w/w dried extract was selected and exhibited an initial pH of 4.43. After 6 months of storage at 4, 30, and 40 °C, the remaining GTG contents were approximately 99.1%, 92.9%, and 87.6%, respectively, indicating temperature-dependent degradation. No microbial contamination was detected during storage. Human closed-patch testing in 30 healthy volunteers yielded an MCII of 0.05, indicating negligible irritation under the tested conditions. Conclusions: These findings support further development of Ya Pra Sa Kra Tung Pew Pai Nok as a standardized topical herbal formulation.