A rapid and versatile HPLC-UVmethod for the separation and quantification of l-citrulline from watermelon was developed for the first time. Freeze dried juice and rind of watermelon were extracted with acidic methanol and analysed by HPLC using 0.03 mM phosphoric acid as a mobile phase. The separation was performed using Zorbax SB-Aq, Synergi Hydro-RP and Gemini C-18 columns and l-citrulline was detected at 207 nm. l-Citrulline was well separated using Gemini C-18 column and the identity of peak was confirmed by mass spectral analysis. These results were further confirmed by derivatisation method. The developed method was used for the quantification of l-citrulline in three varieties of watermelons such as Petite treat, Jamboree and Yellow crimson. Thus, the method is simple and involves direct analysis of aqueous extracts and can be used for rapid routine analysis of commercial samples. In addition, the role of watermelon extracts on smoothmusclerelaxationmarkers was studied. Petite treat and Jamboree extracts induced production of nitric oxide (NO) significantly at 25 ppm. This was also confirmed by measuring the change in the intracellular calcium. Additionally, extracts of petite treat rind and juice have shown inhibition of phosphodiesterase-5A (PDE-5A) in human uterine smoothmuscle cells (UtSMC). The induction of NO and the inhibition of PDE-5A seems to suggest the potency of these extracts to induce relaxation of smoothmuscle. It is possible that watermelon extracts may have the potential to induce PDE-5A mediated smoothmusclerelaxation. However, further studies are critical before any conclusion of smoothmusclerelaxation effect.