This study was carried out to identify some new antidepressant compounds present in Radix Bupleuri (RB) and to develop a method for their quantitative analysis in rat serum for the first time. Four polyacetylenes, including two new compounds, were isolated from Bupleurum scorzonerifoliu and identified. An in vitro uptake study using rat synaptosomes showed that the polyacetylenes potently inhibited serotonin, norepinephrine and dopamine reuptake, and exhibited an antidepressant activity with a potency comparable with or better than their corresponding specific inhibitors. An ultra-performance liquid chromatography coupled with photodiode array detector (UPLC-PDA) method was developed for their quantitative analysis in rat serum. The analysis was performed on a Waters BEH C 18 column (1.7 μm, 100 × 2.1 mm i.d.) using a gradient system of acetonitrile and 0.03% trifluoroacetic acid water, with a detector wavelength of 315 nm. Only two polyacetylenes, ((2Z,8E,10E)-pentadecatriene-4,6-diyn-1-ol (RB-2) and bupleurynol (RB-4)), were detected in the primarily pharmacokinetic study of the petroleum ether fraction of RB. Both were rapidly absorbed and slowly eliminated. The rat exposure was approximately linear under the studied dosages ranging from 22.5 to 90 g/kg herb. In summary, polyacetylenes appear to be the key components responsible for the antidepressant activity of RB, and could be used as chemical standards for the quality evaluation of RB.