Objectives: We performed simultaneous determination of five constituents by HPLC in Samul-tang (SMT). Additionally, we investigated the immune-stimulatory potential of SMT on specific cellular and humoral immune responses in ovalbumin (OVA)-immunized mice. Methods: Reverse-phase chromatography using a Gemini C18 column operating at $40^{circ}C$, and photodiode array (PDA) detection at 190-400 nm, were used for quantification of the five components of SMT. Mobile phase using a gradient flow consisted of two solvent systems. Solvent A was 1.0% (v/v) aqueous acetic acid and solvent B was acetonitrile with 1.0% (v/v) acetic acid. C57BL/6 mice were immunized intraperitoneally with OVA/alum ($100{mu}g/200{mu}g$) on days 1, 8, and 15. The extract of SMT (1000 mg/kg) was given to mice orally for 21 days (from day 1 to day 21). At day 22, OVA-, lipopolysaccharide (LPS)- and concanavalin A (Con A)-stimulated splenocyte proliferation and OVA-specific and total antibodies were measured in plasma. Results: Calibration curves were acquired with $r^2$>0.9999, and the relative standard deviation (RSD, %) for intra- and inter-day precision were both less than 3.5%. The recovery was in the range of 95.69-115.12%, with an RSD less than 6.0%. The contents of five components in SMT were 1.08-15.30 mg/g. SMT significantly enhanced Con A-induced splenocyte proliferation in OVA-immunized mice (p<0.01). Also, SMT significantly enhanced OVAspecific IgG, IgG1 and total IgM levels in plasma compared with the OVA-immunized group. Conclusions: The established method will be applied for the quantification of major components and immunestimulating activity in OVA-immunized mouse model of SMT.