Abstract: Objective To define the optimal conditions for preparing protein chips on aldehyde-coated slides.Methods The proteins were diluted in PBS containing 40% glycerol and spotted on aldehyde-coated slides. After the spots were dried for 1 hour at room temperature, the slides were stored at 4℃. Following block and rinse, the slides were used for immunoassay and the results detected with a scanner. Several key factors that might influence the results were tested, including the number of spots, concentration of protein in the spotting solution, time of immobilization and the blocking reagent.Results Pre-spotting of 10 to 15 spots achieved good homogeneity of the subsequent spots on aldehyde-coated slides. The protein immobilized at 4℃ for 24 to 48 h showed higher fluorescence intensity and clearer images, and the slides blocked with 3% BSA produced the lowest background signal. The concentration of protein in the spotting solution significantly affected the fluorescence intensity.Conclusion To ensure good results in preparing protein chips on aldehyde slides, pre-spotting of 10-15 spots can be necessary followed by immobilization at 4℃ for 24-48h and 3% BSA blocking.