reducing at least one interchain disulfide bond in the activatable antibody with a reducing agent without reducing any intrachain disulfide bonds within a masking moiety (MM) in the activatable antibody, thereby partially reducing the activatable antibody,

wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP),

wherein the ratio of the reducing agent to the activatable antibody is less than 5:1, and

wherein the reduction time is 30 minutes to 2 hours; and

conjugating an agent to at least one thiol of the partially reduced activatable antibody,

wherein the activatable antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target, the MM coupled to the AB that inhibits the binding of the AB to the target when the activatable antibody is in an uncleaved state, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease,

wherein the partial reduction of the activatable antibody and conjugation of the agent thereto does not disturb or negatively affect activation and/or efficacy of the activatable antibody, wherein the activity and/or efficacy of the activatable antibody is selected from the group consisting of: the masking activity of the MM to the AB when the activatable antibody is in an uncleaved state, the activation of the uncleaved activatable antibody by the protease, and the binding activity of the activated activatable antibody to the target of the AB.