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In situ encapsulation is a frequently used method to prepare hydrogels loaded with high quantities of therapeutic proteins. However, many cross-linking reactions, such as Michael-type addition or Diels–Alder (DA) reaction are not tolerant toward nucleophiles; therefore, side-reactions with proteins can occur during cross-linking. This may lead to undesired protein conjugation, activity loss and incomplete protein release. In this study, a number of polyanions, namely alginate, dextran sulfate, hyaluronic acid, heparin, and poly(acrylic acid), were screened for their capability to protect proteins during covalent cross-linking. To this end, lysozyme was incubated with furyl- and maleimide-substituted methoxy poly(ethylene glycol); different pH values were tested. The degree of PEGylation and the residual activity of lysozyme were investigated. Without polyanions, 61.1% of the total lysozyme amount was PEGylated at pH 7.4; the residual activity was 20.3% of the initial activity. With the most effective polyanion (dextran sulfate), PEGylation could be completely suppressed; the residual activity was 98.4%. The protective effect of polyanions was attributed to electrostatic interactions with proteins; the “shielding” could be reversed by adding high salt concentrations. Furthermore, the protective effect was dependent on the concentration and molecular mass of the polyanion, but almost independent of the protein concentration. As a proof of concept, hydrogels were loaded with lysozyme and bevacizumab during cross-linking via DA reaction. Without polyanions, a large fraction of the protein was covalently bound to the polymer network resulting in degradation-controlled release; the residual activity of lysozyme was 50.0%. With polyanions, the protein molecules were mobile and their release was diffusion-controlled. The residual activity of lysozyme was 88.9%; the released bevacizumab was structurally intact. Polyanions can, therefore, be used as protective additive to prevent chemical protein modification during hydrogel cross-linking.