Reducing lipid accumulation in cells and the whole body is critical for the treatment of obesity and type 2 diabetes. However, current anti-diabetic drugs are insufficient either due to the limited efficacy or side effects. This study aimed to identify compounds with potential anti-diabetic properties by reducing lipid accumulation using a biochemical assay screening in 3T3-L1 cells. Our study firstly developed a new method to extract triglyceride (TG, as a marker of cellular lipids) with KOH to avoid the complicated procedures of the conventional chloroform/methanol (C/M) method which hinders its usage as a high throughput (HTP) screening. Our results showed that the presence of KOH used for TG extraction did not influence the enzymatic measurement of TG (Y=1.1416X, R2=0.9978, p<0.001 for the regression of the values measured ± KOH). The extraction of TG with KOH from 3T3-L1 cells was validated by a high recovery rate (90-95%) which was significantly greater (p<0.001) compared with the C/M extraction (12-18%). This novel screening was able to detect the expected changes in TG by different mechanisms including fatty acid (FA) oxidation promoters, FA synthesis inhibitors, PPAR gamma activator and lipogenic substrates. By using this HTP assay, we screened more than 300 compounds from our unique library selected from two traditional Chinese medicine banks and identified 80 hits from 20 classes of small molecules. Five classes of the identified compounds have been demonstrated in our lab with efficacy in reversing insulin resistance or glucose intolerance in vivo. Our study demonstrates that screening for the lipid-lowering efficacy in 3T3-L1 cells is an effective end-point assay capable of capturing potential new anti-diabetic drugs that act at different sites regulating lipid metabolism.