Creatinine, an important biomarker for renal functions, is often conventionally estimated using the gold standard jaffe reaction from blood, which involves the usage of a spectrophotometric measurement, thus restricting its utilization scope in point-of-care settings. here, we report the development of a method for the single-step quantification of creatinine from whole blood using a paper-based microfluidic device. our platform uses whatman filter paper integrated with an lf1 membrane. the on-chip separation of blood plasma is achieved through the lf1 membrane, while the whatman component of the device contains the embedded reagents for the jaffe reaction. the combination of two different grades of paper enables a single-step quantification of creatinine as the separated blood plasma traverses to the reaction zone through capillary imbibition. colorimetric readouts were quantified using an rgb sensor instead of a smartphone, which is highly platform dependent and incurs a relatively higher cost compared to the other components in typical point-of care (poc) devices. our sensor was integrated within a 3d box, thereby making the detection virtually instrument free and perfectly suited for poc settings. the limit of detection (lod) of our device was 0.219 mg dl−1, which falls within the lower range of physiological values. the coefficient of determination (r2) for the linearity and median accuracy were 0.978 and 94.047%, respectively. the relative standard deviation (rsd) for precision measurements remained below 5% for the developed protocol. furthermore, we validated the performance of our device with 35 clinical samples in laboratory settings against the gold standard measurements. our bland–altman plot as well as t-test and chi-square test results clearly confirmed the validity of our device within a 95% confidence interval.