The subfamily Asclepiadoideae (Apocynaceae) and the closely-related Periplocoideae are sources of many indigenous Indian medicinal plants. We surveyed antioxidant properties and total phenolic and flavonoid contents of 15 samples, representing 12 Indian medicinal plant species from these subfamilies. Total antioxidant assay was performed using the 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power methods. Total phenolic and flavonoid contents were measured using colourimetric methods. Principal phenolic compounds were detected by liquid chromatography-mass spectrometry (LC-MS). The highest antioxidant capacity and high levels of total phenolics and flavonoids were found in the leaves of Decalepis hamiltonii. The stems of Sarcostemma brevistigma exhibited the highest xanthine oxidase (XO) inhibitory activity. The roots of Hemidesmus indicus showed the highest OH(-) radical scavenging activity. In general, Periplocoideae members exhibited higher antioxidant activity than Asclepiadoideae members. The highly significant and positive correlations (R > 0.914) between total antioxidant capacity parameters and total phenolic content indicated that the phenolic compounds contributed significantly to the antioxidant activity of the tested plant samples. The principal phenolic phytochemicals from these plants were identified by LC-MS, including flavonoids, phenolic acids and phenolic terpenoids. Chlorogenic acid and rutin were detected in almost all of the plant samples. The LC-MS analysis provided full fingerprints of the principal phenolic compounds in the medicinal plants from these two subfamilies, which are useful for their authentication and quality evaluation.