Surface‐enhanced laser desorption ionization (SELDI) time‐of‐flight mass spectrometry (TOF MS) was used to profile six human ovarian cancer cell lines. Non‐confluent cell cultures were exchanged into serum free medium and allowed to grow for either 24 or 48 hours, at which time the medium was collected and analyzed using a Ciphergen SELDI‐TOF MS and a QSTAR Pulsar QqTOF mass spectrometer fitted with a SELDI source. The spectra showed the presence of several low molecular weight species, as well as differences and similarities in the proteins detected in the media of the six ovarian tumor cell lines. The same species were detected at 24 and 48 hours and reproducible changes in their relative abundances were observed.