Substation protective relay coordination setting plays a vital role in the safe operation of power system. The objective of protective relay coordination in an interconnected power system is to achieve selectivity without sacrificing sensitivity and fast fault clearance time. Proper coordination of relays is essential to minimize unnecessary outages. In recent years, power demand has increase substantially while the expansion of the system has been severely limited due to abnormalities of Isolation of faulty areas by the protection system as a result of lack of effective coordination of the relay operation. This paper presents a study on protection coordination of over-current relays (OCRs) in a 132/33kV transmission substation by considering its different operating modes using a MATLAB model. Over-current (OC) relays are the major protection devices in a distribution system. The operating time of the OC relays are to be coordinated properly to avoid the mal-operation of the backup relays. The purpose is to find optimum values of Time Multiplier Setting (TMS) and Plug Setting (PS) which is used to find optimum coordination of Over-current Relays.