The drug 5-fluorouracil is used to treat several cancers including colon, esophageal, stomach, pancreatic, breast, and cervical cancer. In this study, the solubility of 5-fluorouracil in subcritical water was determined at temperatures ranging from 298 to 473 K under a constant pressure of 5.1 MPa. Our results show that the solubility of 5-fluorouracil is enhanced more than 12 -fold by increasing the temperature from 298 to 473 K. A new approximation model was developed to predict the solubility of 5-fluorouracil in subcritical water. Our predicted solubility values are comparable with the experimental ones. The modified Apelblat equation also yields good agreement with experimental values. Since our solubility study reveals that 5-fluorouracil has adequate solubility in subcritical water, the separation of 5-fluorouradl was conducted using subcritical water chromatography (SBWC) with water only mobile phase. Good chromatographic separation of 5-fluorouracil was achieved at temperatures of 348 to 473 K.