diff --git a/tests/drivers/i2c/i2c_target_api/boards/nrf5340dk_nrf5340_cpuapp.conf b/tests/drivers/i2c/i2c_target_api/boards/nrf5340dk_nrf5340_cpuapp.conf
new file mode 100644
index 00000000000..935827e4233
--- /dev/null
+++ b/tests/drivers/i2c/i2c_target_api/boards/nrf5340dk_nrf5340_cpuapp.conf
@@ -0,0 +1,7 @@
+# Copyright (c) 2024 Nordic Semiconductor ASA
+# SPDX-License-Identifier: Apache-2.0
+
+CONFIG_I2C_VIRTUAL=n
+CONFIG_BOOT_BANNER=n
+CONFIG_I2C_NRFX_TWIS_BUF_SIZE=256
+CONFIG_I2C_TARGET=y
diff --git a/tests/drivers/i2c/i2c_target_api/boards/nrf5340dk_nrf5340_cpuapp.overlay b/tests/drivers/i2c/i2c_target_api/boards/nrf5340dk_nrf5340_cpuapp.overlay
new file mode 100644
index 00000000000..42bd62733b3
--- /dev/null
+++ b/tests/drivers/i2c/i2c_target_api/boards/nrf5340dk_nrf5340_cpuapp.overlay
@@ -0,0 +1,50 @@
+/*
+ * Copyright (c) 2024 Nordic Semiconductor ASA
+ *
+ * SPDX-License-Identifier: Apache-2.0
+ */
+
+&pinctrl {
+	i2c2_default: i2c2_default {
+		group1 {
+			psels = <NRF_PSEL(TWIS_SDA, 0, 26)>,
+				<NRF_PSEL(TWIS_SCL, 0, 25)>;
+			bias-pull-up;
+		};
+	};
+
+	i2c2_sleep: i2c2_sleep {
+		group1 {
+			psels = <NRF_PSEL(TWIS_SDA, 0, 26)>,
+				<NRF_PSEL(TWIS_SCL, 0, 25)>;
+			low-power-enable;
+		};
+	};
+};
+
+&i2c2 {
+	compatible = "nordic,nrf-twis";
+	pinctrl-0 = <&i2c2_default>;
+	pinctrl-1 = <&i2c2_sleep>;
+	pinctrl-names = "default", "sleep";
+	status = "okay";
+
+	eeprom0: eeprom@54 {
+		compatible = "zephyr,i2c-target-eeprom";
+		reg = <0x54>;
+		address-width = <8>;
+		size = <256>;
+	};
+};
+
+&i2c1 {
+	zephyr,concat-buf-size = <256>;
+	status = "okay";
+
+	eeprom1: eeprom@56 {
+		compatible = "zephyr,i2c-target-eeprom";
+		reg = <0x56>;
+		address-width = <8>;
+		size = <256>;
+	};
+};