The aim of this research was to evaluate bromelain from pineapple stem in cream based preparation and to observe in vitro anti-inflammatory activity of the cream. Optimization of three different cream formulas were done to determine the most stable one. During four weeks of storage period, formula I with a mixture of Olivem 1000 and Tween 60 surfactants had the best physical form and emulsion stability compared to the other two formulas, with a pH value at 27°C of 5.40 ± 0.00 and 5.71 ± 0.008 at week 0 and week 4 respectively. The bromelain used as active substance in formula I cream preparation was isolated by partial purification of pineapple stem extract (Ananas comosus [L.] Merr) by fractionation method using ammonium sulfate followed by dialysis, with a specific activity of 155.58 U/mg. Evaluation results of bromelain activity in optimized cream preparation showed an enzyme activity of 0.52 U/mL. Penetration test for bromelain cream was done based on the Franz Diffusion Cell method. The results showed that in 8 hours, the cumulative penetrated amount of bromelain in cream preparation was 793.45 μg/cm2. The anti-inflammatory test using the HRBC method for cream preparation containing each commercial bromelain, isolated bromelain, and aspirin with the same concentration (2%), showed percent stability results of 34.41%, 32.06%, and 37.82% respectively. Thus, it could be concluded that cream preparations containing bromelain from pineapple stem have moderate anti-inflammatory activity.