Cell suspension and Agrobacterium rhizogenes-transformed hairy root cultures of Devil’s claw (Harpagophytum procumbens), an African plant with high medicinal value, were cultivated in shake-flasks. A purification scheme to isolate their main active constituents (the phenylethanoid glycosides verbascoside, leucosceptoside A, β-OH-verbascoside and martynoside; structurally identified by NMR and LC–MS) was then developed, and their concentrations in the cultures were determined by UV spectrometry following HPLC separation. Preparations, extracts and the isolated phenylethanoid glycosides from the H. procumbens in vitro systems were tested on isolated murine macrophages to study their effects on nitric oxide (NO) and cytokine (TNF-α, IL-6) release and the expression of COX-1 and COX-2. They were also added to human serum to investigate their effects on the classical pathway of complement activation. The results indicate that the extracts and preparations of the in vitro systems, and pure verbascoside (their main active constituent), had strong anti-inflammatory properties, comparable to or even higher than that of pure harpagoside (a major anti-inflammatory constituent of intact Devil’s claw tubers). Thus, they have potential as new anti-inflammatory agents.