diff --git a/tests/drivers/i2c/i2c_target_api/boards/nrf54l20pdk_nrf54l20_cpuapp.conf b/tests/drivers/i2c/i2c_target_api/boards/nrf54l20pdk_nrf54l20_cpuapp.conf
new file mode 100644
index 00000000000..2510d6673c1
--- /dev/null
+++ b/tests/drivers/i2c/i2c_target_api/boards/nrf54l20pdk_nrf54l20_cpuapp.conf
@@ -0,0 +1 @@
+CONFIG_I2C_NRFX_TWIS_BUF_SIZE=256
diff --git a/tests/drivers/i2c/i2c_target_api/boards/nrf54l20pdk_nrf54l20_cpuapp.overlay b/tests/drivers/i2c/i2c_target_api/boards/nrf54l20pdk_nrf54l20_cpuapp.overlay
new file mode 100644
index 00000000000..f04022cf8f3
--- /dev/null
+++ b/tests/drivers/i2c/i2c_target_api/boards/nrf54l20pdk_nrf54l20_cpuapp.overlay
@@ -0,0 +1,68 @@
+/*
+ * SDA = P1.8 and P1.9
+ * SCL = P1.14 and P1.15
+ */
+
+&pinctrl {
+	i2c21_default: i2c21_default {
+		group1 {
+			psels = <NRF_PSEL(TWIS_SDA, 1, 8)>,
+				<NRF_PSEL(TWIS_SCL, 1, 14)>;
+			bias-pull-up;
+		};
+	};
+
+	i2c21_sleep: i2c21_sleep {
+		group1 {
+			psels = <NRF_PSEL(TWIS_SDA, 1, 8)>,
+				<NRF_PSEL(TWIS_SCL, 1, 14)>;
+			low-power-enable;
+		};
+	};
+
+	i2c22_default: i2c22_default {
+		group1 {
+			psels = <NRF_PSEL(TWIS_SDA, 1, 9)>,
+				<NRF_PSEL(TWIS_SCL, 1, 15)>;
+			bias-pull-up;
+		};
+	};
+
+	i2c22_sleep: i2c22_sleep {
+		group1 {
+			psels = <NRF_PSEL(TWIS_SDA, 1, 9)>,
+				<NRF_PSEL(TWIS_SCL, 1, 15)>;
+			low-power-enable;
+		};
+	};
+};
+
+&i2c21 {
+	pinctrl-0 = <&i2c21_default>;
+	pinctrl-1 = <&i2c21_sleep>;
+	pinctrl-names = "default", "sleep";
+	zephyr,concat-buf-size = <256>;
+	status = "okay";
+
+	eeprom1: eeprom@56 {
+		compatible = "zephyr,i2c-target-eeprom";
+		reg = <0x56>;
+		address-width = <8>;
+		size = <256>;
+	};
+};
+
+&i2c22 {
+	compatible = "nordic,nrf-twis";
+	pinctrl-0 = <&i2c22_default>;
+	pinctrl-1 = <&i2c22_sleep>;
+	pinctrl-names = "default", "sleep";
+	status = "okay";
+
+	eeprom0: eeprom@54 {
+		compatible = "zephyr,i2c-target-eeprom";
+		reg = <0x54>;
+		address-width = <8>;
+		size = <256>;
+	};
+};
diff --git a/tests/drivers/i2c/i2c_target_api/testcase.yaml b/tests/drivers/i2c/i2c_target_api/testcase.yaml
index 588b7f7a43d..4237b60b2dc 100644
--- a/tests/drivers/i2c/i2c_target_api/testcase.yaml
+++ b/tests/drivers/i2c/i2c_target_api/testcase.yaml
@@ -57,5 +57,6 @@ tests:
       - nrf5340dk/nrf5340/cpuapp
       - nrf54h20dk/nrf54h20/cpuapp
       - nrf54l15dk/nrf54l15/cpuapp
+      - nrf54l20pdk/nrf54l20/cpuapp
     integration_platforms:
       - max32690evkit/max32690/m4