Dialysis is the rate-limiting step in the preparation of aqueous silk fibroin (SF) solution. However, the traditional practice of dialyzing SF solution for at least 48 h to remove LiBr is not based on empirical evidence. The aim of the present study was to systematically measure LiBr content in SF solutions dialyzed for varying lengths of time and assess the potential toxicity of residual lithium ions in cells. Complete removal of lithium ions was not achieved even after 72 h of dialysis, with a residual lithium ion content in the solution of 22.85 mg/l. SF films prepared from solutions dialyzed for 8 and 24 h had predominantly random coil or b-sheet structures, respectively. The residual lithium had little cytotoxicity in NIH3T3 fibroblast cells, but viability was compromised in cells grown on SF film prepared from solution dialyzed for 24 h.