What exactly do you mean by 'cell free filtrate' from your protozoa? It sounds like you want to prepare a lysate from those cells and you want to purify out bacteria before applying it to some other cell line to asses toxicity. 

If that is the case, perhaps you could leverage a size disparity between the bacterial feeder cells and the protozoa. For example you would grow up the culture as you normally do, and then pass through a filter which allows bacteria to pass but traps the protozoa. Then apply your lysis solution directly to the filter and collect the flow-through.

Not the lysate, I mean the secreted proteins by the protozoa into the media. Lysate would mean opening them up which I don't want. This is the reason its a bit difficult because there seems to be no way around to exclude the bacterial secreted proteins. On top of this, the bacterial secretome had effect on the monolayer. I am now looking for an assay to differentiate or perhaps quantify this effect between the two. 

I'm just speculating, but will the protozoa tolerate killed bacteria as a background? E.g. you could heat-treat bacteria (or expose them to O2 if they're obligate anaerobes) prior to using them in your media. At least that would help you eliminate active bacterial secretions, though you'd have to be careful that you don't end up rupturing them and ending up with bacterial lysate in your media.