AAT Bioquest

PhosphoWorks™ Colorimetric ATP Assay Kit

ATP dose response measured with PhosphoWorks™ Colorimetric ATP Assay Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices).
ATP dose response measured with PhosphoWorks™ Colorimetric ATP Assay Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices).
ATP dose response measured with PhosphoWorks™ Colorimetric ATP Assay Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices).
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Adenosine triphosphate (ATP) plays a fundamental role in cellular energetics, metabolic regulation and cellular signaling. It is referred as the \"molecular unit of currency\" of intracellular energy transfer to drive many processes and chemical synthesis in living cells. ATP also serves as a signaling molecule for cell communication and plays an important role in DNA and RNA synthesis. AAT Bioquest offers a variety of bioluminescence assay kits to determine nanomolar (nM) range of ATP with recombinant firefly luciferase (Cat# 21610 & 21609). These kits require luminescence plate readers, are frequently used for cell viability or cytotoxicity assays. PhosphoWorks™ Colorimetric ATP Assay Kit is based on a serial ATP-induced enzyme coupled reactions to produce hydrogen peroxide, which is spectrophotometrically quantified with our Amplite® Red Substrate at OD 570 nm. The assay can detect ~3 µM of ATP in a 100 µL reaction volume with minimal interference from ADP and AMP. It provides a robust, simple and convenient assay for measuring ATP levels in biological samples. The PhosphoWorks™ Colorimetric ATP Assay is complementary to our luciferase-based ATP assay kits.


Absorbance microplate reader

Absorbance570 nm
Recommended plateClear bottom


Example protocol


Protocol summary

  1. Prepare ATP working solution (50 µL)
  2. Add ATP standards or test samples (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Monitor absorbance at 570 nm

Important notes
Thaw the kit components at room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. AmpliteTM Red Substrate stock solution (200X):
Add 30 µL of DMSO (Component E) into the vial of AmpliteTM Red Substrate (Component A) to make 200X AmpliteTM Red Substrate stock solution.

2. ATP standard solution (10 mM):
Add 0.5 mL of ddH2O into the vial of ATP Standard (Component D) to make 10 mM ATP standard solution.


ATP standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21617

Add 10 µL of 10 mM ATP standard solution into 990 µL 1X PBS buffer to generate 100 µM ATP standard solution (AS7). Take 100 µM ATP standard solution (AS7) and perform 1:2 serial dilutions to get serially diluted ATP standards (AS6-AS1) with 1X PBS buffer.


1. Add 5 mL of Assay Buffer (Component C) into Enzyme Mix bottle (Component B), and mix well.

2. Add 25 µL of 200X AmpliteTM Red Substrate stock solution to the Enzyme Mix bottle, and mix well to make ATP working solution.


Table 1. Layout of ATP standards and test samples in a clear bottom 96-well microplate. AS= ATP Standards (AS1 - AS7, 1.56 to 100 µM), BL=Blank Control, TS=Test Samples. 


Table 2. Reagent composition for each well. 

AS1 - AS750 µLSerial Dilutions (1.56 to 100 µM) 
BL50 µL1 X PBS Buffer 
TS50 µLTest Sample
  1. Prepare ATP standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of ATP working solution to each well of ATP standard, blank control, and test samples to make the total ATP assay volume of 100 µL/well. For a 384-well plate, add 25 µL of ATP working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.

  4. Monitor the absorbance increase with an absorbance plate reader at OD of 570 nm.



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