Halogen　bonding　(or　X　bonding)　has　attracted　increasing　interest　due　to　its　significant　role　in　molecular　recognition　in　biological　systems.　Trypsin-like　serine　proteases　have　many　physiological　and　pathophysiological　functions.　There　is　therefore　extensive　interest　in　generating　specific　inhibitors　for　pharmacological　intervention　in　their　enzymatic　activity.　We　study　here　if　it　is　possible　to　use　halogenated　compounds　as　the　P1　group　to　bind　to　the　S1　specificity　pocket　of　trypsin-like　serine　proteases　to　avoid　the　low　bioavailability　of　the　amidine　or　guanidine　P1　group　that　is　typically　used　in　many　inhibitors.　We　used　4-chlorobenzylamine　(ClBA),　4-bromobenzylamine　(BrBA)　and　4-iodobenzylamine　(IBA)　as　probes　to　test　their　binding　modes　to　a　trypsin-like　serine　protease,　urokinasetype　plasminogen　activator　(uPA),　which　has　been　recognized　as　a　marker　for　breast　cancer　and　an　important　target　for　inhibitor　development.　The　results　showed　that　these　compounds　inhibited　uPA　with　stronger　efficacies　compared　with　their　non-halogenated　analogues.　We　also　determined　the　highresolution　crystal　structures　of　uPA　in　complex　with　BrBA　and　IBA,　respectively.　The　structures　revealed　that　BrBA　bound　to　the　S1　pocket　of　uPA　via　halogen　bonds,　but　IBA　did　not　make　halogen　bonds　with　uPA,　demonstrating　that　the　iodine　may　not　be　the　best　choice　as　a　target　moiety　for　serine　proteases.　These　results　advocate　halogen　bonding,　especially　bromine　bonding,　as　an　efficient　strategy　for　the　future　design　of　novel　inhibitors　against　trypsin-like　serine　proteases　to　provide　strong　potency　and　promote　bioavailability.