Background: The spot urine protein/creatinine ratio (UPCR) is proposed to be a substitute for 24-hour urinary protein (24h-UP). This study is aimed to determine whether the predictive accuracy of 24h-UP using UPCR can be improved by simply multiplying estimated daily urine creatinine excretion (eUCr) and UPCR together. Methods: This study enrolled 120 participants to investigate the correlation between spot UPCR and 24h-UP. Three sets of spot urine samples were randomly collected throughout the day and night, along with the first morning void. UPCR was weighted by eUCr to investigate the improvement of accuracy in using spot urine samples to predict 24h-UP. Results: There were strong correlation and concordance between UPCR and 24h-UP irrespective of the time of spot urine sampling, and the correlation, concordance and agreement were improved after multiplying the UPCR value by the eUCr. Greater improvement was found in the subgroups with measured daily urine creatinine excretion ≤0.8 g/d and ≥1.2 g/d. Conclusions: This investigation demonstrated that multiplying UPCR by eUCr can improve the accuracy of only using UPCR to predict 24h-UP.